Bioaerosol detection systems and methods of use

ABSTRACT

Described herein are bioaerosol detection systems and methods of use.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the 35 U.S.C. § 371 national stage application ofPCT Application No. PCT/US2017/026305, filed Apr. 6, 2017, which claimspriority to and benefit of U.S. Provisional Application entitled“BIOAEROSOL DETECTION SYSTEMS AND METHODS OF USE,” having Ser. No.62/318,962, filed on Apr. 6, 2016, both of which are entirelyincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberDBI-1353423 awarded by the National Science Foundation. The governmenthas certain rights to the invention.

BACKGROUND

Existing bioaerosol sampling systems are designed for bacterial aerosolcollection/detection based on inertia principles. They are veryineffective for viral aerosols because viruses are much smaller thanbacteria and are not effectively captured by the equipment. In additionto being ineffective, conventional detection methods for viruses aretime consuming and do not allow determination in field investigation.The present disclosure discusses systems and methods for bioaerosoldetection, in particular viral aerosols, to address the aforementioneddeficiencies and inadequacies.

SUMMARY

Described herein are embodiments of bioaerosol amplification anddetection systems. Bioaerosol amplification and detection systems asdescribed herein can be modular systems and can comprise a bioaerosolamplification unit comprising a chamber configured to receive aircontaining bioaerosols, wherein the chamber is further configured foradiabatic amplification of the bioaerosols; and a biosampler, whereinthe biosampler is in fluid communication with the bioaerosolamplification unit, and wherein the biosampler is configured to receiveand collect adiabatically amplified bioaerosols from the chamber of thebioaerosol amplification unit.

Additionally, embodiments of bioaerosol amplification and detectionsystems described herein can further comprise adiabatic amplification byadiabatic cooling. In certain embodiments, adiabatic cooling can furthercomprise swirling, mixing, or both, of air containing bioaerosols.

In certain embodiments, bioaerosol amplification and detection systemsas described herein, can further comprise a bioaerosol analysisplatform, wherein the bioaerosol analysis platform is configured toreceive adiabatically amplified bioaerols collected in the biosamplerand configured to detect the adiabatically amplified bioaerosols by oneor more bioaerosol detection assays. In certain embodiments, thebioaerosol analysis platform can be a microfluidic device comprising oneor more bioaerosol detection assays configured to detect theadiabatically amplified bioaerosols.

In certain embodiments, bioaerosol amplification units as describedherein can further comprise one or more interior surfaces of the chamberwetted with water having a temperature of about 35° C. to about 65° C.,wherein the one or more surfaces is adjacent to the air containingbioaerosols.

In certain embodiments, the chamber further comprises cooled aircontaining bioaerosols having a temperature of about −40° C. to about10° C. and steam having a temperature of about 35° C. to about 65° C. Incertain embodiments, the chamber further comprises cooled air having aflow rate of about 0.1 Liters/min to about 10 Liters/min and steamhaving a flow rate of about 1 Liters/min to about 50 Liters/min.

In certain embodiments, the air containing bioaerosols can be cooled bya temperature drop within the chamber of the bioaerosol amplificationunit, wherein the temperature drop is controlled by the ratio of thepressure of the air containing bioaerosols after adiabatic expansion tothe pressure of the air containing bioaerosols before adiabaticexpansion.

In certain embodiments, microfluidic devices of systems as describedherein can be paper-based or laminated paper-based. In certainembodiments, one or more detection assays of systems described hereincan comprise an immunoassay or a nucleic acid amplification assay,individually or in combination. In certain embodiments, bioaerosolamplification and detection systems as described herein can beconfigured to detect viruses.

Also described herein are methods of detecting amplified bioaerosols.Methods as described herein can comprise the steps of: providing abioaerosol amplification and detection system comprising a bioaerosolamplification unit, a biosampler, and a bioaerosol analysis platform;delivering air containing bioaerosols to the bioaerosol amplificationunit, wherein the bioaerosol amplification unit is configured toadiabatically amplify bioaerosols; adiabatically amplifying bioaerosolswith the bioaerosol amplification unit; delivering amplified bioaerosolsfrom the bioaerosol amplification unit to the biosampler; precipitating,concentrating, or both the amplified bioaerosols into a collectionreservoir of the biosampler; delivering the collected amplifiedbioaerosols from the collection reservoir of the biosampler to abioaerosol analysis platform, wherein the bioaerosol analysis platformis configured to detect one or more collected amplified bioaerosols orcomponents thereof with one or more detection assays; and detectingcollected amplified bioaerosols or bioaerosol components with one ormore detection assays. Adiabatic amplification in methods as describedherein can be adiabatic cooling, and in certain embodiments can includeswirling and mixing of air containing bioaerosols.

In certain embodiments of methods as described herein, one or moredetection assays used in the methods can be one or more nucleic aciddetection assays or one or more immunoassays, individually or incombination and the assays can be configured to detect one or moreviruses.

In certain embodiments of methods as described herein the biosampler canfurther comprise a collection media. In certain embodiments of methodsdescribed herein, the air containing bioaerosols is cooled by atemperature drop within the bioaerosol amplification unit, wherein thetemperature drop is controlled by the ratio of the pressure of the aircontaining bioaerosols after adiabatic expansion to the pressure of theair containing bioaerosols before adiabatic expansion.

In certain embodiments of methods as described herein the air containingbioaerosols can be cooled within a chamber of the bioaerosolamplification unit, wherein the chamber of the bioaerosol amplificationunit has one or more interior surfaces adjacent to the air containingbioaerosols, wherein the one or more surfaces are wetted with warmwater.

In certain embodiments of methods as described herein the chamber can beconfigured so that the volume of the chamber can be reduced bycompression and expanded by decompression.

In certain embodiments of methods as described herein the biosampler canbe functionally integrated into the chamber of the bioaerosolamplification unit and the chamber is configured for collection ofamplified bioaerosols.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the disclosed devices and methods can be betterunderstood with reference to the following drawings. The components inthe drawings are not necessarily to scale, emphasis instead being placedupon clearly illustrating the relevant principles. Moreover, in thedrawings, like reference numerals designate corresponding partsthroughout the several views.

FIGS. 1(a)-(b) illustrates traditional bioaerosol capture principles.

FIG. 2 shows collection efficiency of traditional bioaerosol samplers asa function of particle diameter.

FIGS. 3(a)-(e) demonstrates a fabrication process for a LaminatedPaper-based Analytical Device (LPAD).

FIGS. 4(a)-(b) illustrate: a) a diagram of a bioaerosol amplificationunit (BAU) together as described herein in fluid communication with b) abiosampler as described herein.

FIGS. 5(a)-(b) shows how adiabatic cooling can be used to createsupersaturation.

FIGS. 6(a)-(b) show different mixing ratios between aerosol flow andsteam.

FIGS. 7(a)-(b) demonstrates a representative design of a paper-basedanalytical device.

FIG. 8 shows a microfluidic device for improving throughput volume.

FIG. 9 illustrates an integrated system for the collection of lowconcentration viral aerosols.

FIGS. 10(a)-(b) demonstrates an embodiment of an experimental systemthat can be used for testing the effectiveness of the BAU and an entireintegrated system.

FIG. 11 shows MS2 aerosol particle size distribution with and withoutadiabatic expansion as described herein.

FIG. 12 illustrates concentration of viable MS2 viruses collected withand without adiabatic expansion as described herein.

FIGS. 13(a)-(g) shows an embodiment of a paper-based microfluidic deviceaccording to the present disclosure and a method of fabrication.

FIGS. 14(a)-(b) demonstrates an embodiment of device preparation and achemiluminescence-based immunoassay protocol for sample detectionaccording to the present disclosure.

FIGS. 15(a)-(d) show bioanalyzer results of a typical nucleic acidsequence-based amplification (NASBA) reaction showing MS2 and flu viralamplicons.

FIGS. 16(a)-(d) shows typical flu virus reverse transcriptionloop-meditated isothermal amplification (RT-LAMP) reactions andcolorimetric detection thereof.

FIG. 17 demonstrates an exemplary scheme for viral detection usingNASBA. The NASBA reaction can be carried out in a centrifugal collectiontube with the assistance of magnetic beads and a magnetic collectiontube holder.

FIG. 18(a)-(b) illustrates an embodiment of an immiscible phaseseparation device that can be used to carry out detection assays such asan enzyme-linked immunosorbent assay (ELISA) in a point-of-care format.

FIGS. 19(a)-(f) demonstrates an embodiment of an LPAD, which can bedesigned and configured to perform virus lysis, RNA extraction, andRT-LAMP detection all together in one device.

FIGS. 20(a)-(d) show an exemplary scheme for RT-LAMP and colorimetricdetection based on the embodiment of the LPAD of FIGS. 19(a)-(f).

FIGS. 21(a)-(b) illustrate an embodiment of an LPAD device for virusdetection, such as nucleic acid virus detection. FIG. 21(a) shows layersand construction of a device and FIG. 21(b) is a photograph depicting anembodiment of a device.

FIGS. 22(a)-(b) shows an embodiment of detection of H1N1 flu virus RNAusing the device of FIG. 21 verified by agarose gel staining. The labelsabove the lanes mark the TCID50 number of flu virus tested in thedevice, and the “−” marks the negative control.

FIGS. 23(a)-(d) demonstrate an embodiment of in-device colorimetric H1N1flu virus detection using phenol red and SYBR Green I DYE and the deviceof FIG. 21. “+” marks sample[s] positive for H1N1 and “−” marks samplesnegative for H1N1. FIG. 23(a) is a photograph showing two devices withphenol red RT-LAMP buffer before incubation. FIG. 23(b) shows thedevices of FIG. 23(a) after incubation, positive sample device turns toorange (left) from pink (right). FIG. 23(c) shows the RT-LAMP bufferincubated in the device of FIGS. 23(a) and 23(b) under ambient lightafter adding SYBR Green I dye. FIG. 23(d) shows the two samples of FIG.23(c) observed under a blue LED flashlight. The positive sample (left)has a green fluorescence.

FIGS. 24(a)-(d) show a schematic diagram of an embodiment of a BADS (aBatch Adiabatic-expansion for Size Intensification by Condensation, orBASIC), sampler, its operation, and the experimental system setup.

FIG. 25. shows Particle size distribution of aerosol from BASIC incomparison with source aerosol, without adiabatic expansion and withadiabatic expansion at different compression pressures.

FIGS. 26(a)-(b) illustrate a comparison of particle number concentrationand CMD of supermicron particles under different compression pressurelevels.

FIGS. 27(a)-(b) illustrate a comparison of particle number concentrationand CMD of supermicron particles under 3 numbers of C/E cycles.

FIGS. 28(a)-(b) show a comparison of particle number concentration andCMD of supermicron particle under 3 water temperature levels.

FIGS. 29(a)-(d) demonstrate a comparison of MS2 titer in the BASICsampler with variation of four factors.

FIG. 30 is a table showing experimental design of sensitivity analyseson physical size amplification and viability preservation.

FIG. 31 is a table showing number concentration and count median (CMD)of supermicron particles in the air sample from BASIC, with and withoutadiabatic expansion.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit (unlessthe context clearly dictates otherwise), between the upper and lowerlimits of that range, and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of chemistry, inorganic chemistry, materialscience, and the like, which are within the skill of the art. Suchtechniques are explained fully in the literature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is inatmosphere. Standard temperature and pressure are defined as 25° C. and1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

Discussion

Embodiments of the present disclosure provide for bioaerosolamplification and detection systems, bioaerosol analysis platforms,methods of using bioaerosol analysis platforms, bioaerosol amplificationunits, methods of using bioaerosol amplification units, biosamplers,methods of using biosamplers, method of detecting amplified aerosols,and the like. Embodiments of the present disclosure provide for highlyefficient and rapid bioaerosol (e.g., viral bioaerosol) detection, ordetection of bioaerosol components (e.g., nucleic acids, proteins,lipids, carbohydrates, or other surface or non-surface antigens).

Monitoring and detection of airborne bioaerosols is of great importance.There is especially a need for unattended monitoring of airborne virusesin the ambient air and in-situ assessment of contaminated locations allcall for development of a rapid bioaerosol detection instrument that iscapable of functioning effectively outside the controlled laboratoryconditions. Such a capability will be instrumental in protecting thehealth and security of humans in military camps, public areas, schools,hospitals, airports, conflict zones and so on.

Embodiments of the present disclosure can have applications in publichealth, environmental health and medicine studies. It will facilitatereal-time indoor air quality sampling in critical environments such ashospitals, clinics, emergency and clean rooms. It will also helpdiagnosing viral aerosol existing inside the exhaled breath.

Traditional air sampling and monitoring systems and methods can beinefficient, inaccurate, and unsuitable when it comes to detecting andanalyzing small bioaerosols on the nano-meter scale. There is a need forimproved devices and methodology.

In general, embodiments of the present disclosure are directed to abioaerosol amplification and detection system (BADS). BADS of thepresent disclosure can comprise one or more modules, for example abioaerosol amplification unit (BAU), a biosampler, and/or a bioaerosolanalysis platform (BAP). In certain embodiments, a BADS can comprise aBAU and a biosampler. In certain embodiments, a BADS can comprise a BAU,a biosampler, and a BAP. In certain embodiments, two or more modules canbe integrated into a single module, for example a module can be presentwhich is comprised of an integrated BAU and biosampler.

One skilled in the art will recognize that the modular systems describedherein can be realized through various combinations and configurationsof modules, and that modules with different functions can be integrated,and embodiments of BADS as described herein should not be construed aslimited to configurations only as described herein. Further, althoughmuch discussion is directed towards bioaerosols, it will be readilyapparent to one skilled in the art that devices and methods as describedherein can be applied to the amplification and detection ofnon-biological, organic or inorganic aerosols as well.

An embodiment of the present amplified bioaerosol detection system usesa BAU that can utilize adiabatic cooling/expansion, swirling/mixing, andwetted walls in combination to amplify bioaerosols. Swirling, mixing, orboth can be optional and may not be necessary for efficient adiabaticamplification by adiabatic cooling/expansion. Swirling/mixing mayimprove amplification efficiency in certain embodiments. Wetted wallscan also be an optional feature of systems described herein. In certainembodiments, adiabatic cooling/expansion can be accomplished withcompression/decompression of air containing bioaerosols.

In an embodiment of the present BADS, air containing bioaerosols can bedrawn into the BAU and then amplified. After amplification by the BAUwithin the amplified aerosol detection system, bioaerosols can be sentto a collection reservoir in a biosampler that collects amplifiedbioaerosol particles. The collection reservoir within the biosampler ofthe BADS can contain a collection media/medium that can preservebioaerosols, amplified bioaerosols, or components thereof suitable fordetection and/or analysis. The BADS or components thereof can bemodified to improve concentration of collected bioaerosols if needed. Adevice, such as an impinger or electrostatic precipitator for example,can be used to aid collection if necessary within the biosampler of theBADS.

Bioaerosols as described herein can be a fungi, bacteria, mycotoxin, orvirus or groupings thereof. A bioaerosol can be a fungal cell such as aspore, mold, and/or yeast, and can be active or inactive. A bioaerosolcan be a gram-positive or gram-negative bacteria and can be a rod-,sphere-, or spiral-shaped prokaryote. A bacterial bioaerosol can be asmall bacteria of sub-micrometer size such as a bacteria of the genusMycoplasma or an otherwise ultramicrobacteria. In an embodiment, thebioaerosol can be a virus. A viral bioaerosol can be a DNA or an RNAvirus, and can have a genome, a capsid, and optionally an envelope. Thegenome can include a single stranded RNA or DNA or double stranded RNAor DNA. The genome can be positive sense or negative sense if it issingle stranded. A bioaerosol can be an MS2 bacteriophage or aninfluenza virus. A viral bioaerosol can have a variety of shapes, andthe shape of the viral bioaerosol should be construed as limiting withregards to the discussion herein. Bioaerosol structure and function canbe conveyed with components such as proteins, nucleic acids, lipids,and/or carbohydrates for example. These components can be detected bydetection assays. Components of bioaerosols that can be detected can bepresent on the bioaerosol surface, linked to the surface, in theinterior of a bioaerosol (inside a capsid, wall, or membrane), in anenvelope, or can be a constituent of a capsid, cell wall, envelope ormembrane for example. Bioaerosol lysis can be used to detect interiorbioaerosol components that reside within a capsid, cell wall, membrane,or envelope. Air containing bioaerosols may contain non-biologicalaerosols, which may be amplified by the present system.

After amplified bioaerosols are collected in the biosampler of the BADS,the BADS can use a BAP to detect bioaerosols or components thereof.Embodiments of the BAP incorporate nucleic acid detection assays and/orimmunoassays into the BADS to detect bioaerosols, such as viruses, orbioaerosol components (proteins, nucleic acids, etc). Embodiments of theBAP of the BADS can use a device for bioaerosol detection. The devicecan be a microfluidic device, which are excellent platforms fordetection and/or analysis in part because of the low sample volumes theyrequire. Paper-based microfluidic devices are especially useful becausethey are inexpensive and easy to construct. Embodiments of the presentBADS use a paper-based microfluidic device within the BAP for bioaerosoldetection. A lamination procedure can be used to laminate thepaper-based microfluidic device in the system to improve mechanicalstrength of the device, and in an embodiment the BAP of the BADS uses alaminated paper-based analytical device (LPAD) or laminated paper-basedmicrofluidic device for bioaerosol detection. An LPAD can be a laminatedpaper-based microfluidic device. Any number of detection methods can becoupled to the microfluidic device of the BAP in the present BADS todetect amplified bioaerosols, and one skilled in the art would recognizewhich detection methods are suitable for detection and/or analysisdepending on the desired application.

In an embodiment the system can contain a bioaerosol amplification unit(BAU). The bioaerosol amplification unit can have a chamber in which aircontaining bioaerosols is drawn into. In certain embodiments, thechamber can be compressible. The chamber can have one or more walls thatcan be covered with a wick and wetted with a liquid, such as water. Theliquid used to the wet the wick on the chamber wall[s] can be waterhaving a temperature of about 35° C. to about 65° C. The wick can beporous and hydrophilic. Water can spread throughout the wick usingcapillary action and/or gravity. Excess water on the walls of thechamber and/or wick can be drained with a drain. A filter can be placedon the drain. In an embodiment, the liquid on the chamber wall is warmand can be water. In an embodiment, the liquid throughout the wick iswarm and can be water. The wick can stay wet by pumping liquid to thewick with a pump.

In an embodiment, air to be sampled can be drawn into the chamber of theBAU with a vacuum pump. The air can contain aerosols, especiallybioaerosols. Viral particles (also described herein as viral aerosols orviral bioaerosols) can be among the bioaerosols in the air. Thebioaerosol-containing air can be cooled. In an embodiment, thebioaerosol-containing air is cooled with adiabatic cooling within theBAU. The cool bioaerosol-containing air can be mixed with warm steam toinduce swirling-mixing. In an embodiment, the mixing ratio (warm steamflow rate/cold aerosol flow rate) can be varied to controlswirling-mixing behavior of the aerosol and the warm steam. In anembodiment, the mixing ratio (warm steam flow rate/cold aerosol flowrate) is high. In an embodiment, the mixing ratio can be controlled witha controller. In an embodiment, a temperature gradient exists betweenthe wetted walls of the chamber and the warm steam. In an embodiment,the chamber of the BAU can be configured to produce or accommodate atemperature drop, and the temperature drop can be controlled by theratio of the pressure of the air within the chamber after expansion tothe ratio of the air within the chamber before expansion. The air canoptionally be compressed within the chamber to aid in amplification. Thebioaerosol-containing air can be mixed with swirling-mixing. In anembodiment, swirling-mixing is induced by mixing cooledbioaerosol-containing air with warm steam. The bioaerosols in the aircan be amplified in combination with water vapor condensation. In anembodiment, water vapor condenses on the bioaerosols in the air toamplify particle size. In an embodiment, the air contains viralparticles or bioaerosols or aerosols that are amplified by the BAU.Viral particles can be amplified or enlarged from nanometer-sized tomicrometer-sized. In an embodiment, the BAU is a viral amplificationunit (VAU) that amplifies viral aerosols.

The bioaerosol containing air within the BAU can have a temperature ofabout −40° C. to about 10° C., about −30° C. to about 0° C., or about−20° C. to about −10° C. Bioaerosol-containing air within the BAU canhave a flow rate of about 1 Liters/min to about 10 Liters/min, about 2Liters/min to about 9 Liters/min, about 3 Liters/min to about 8, about 4Liters/min to about 7 Liters/min Liters/min, or about 5 Liters/min toabout 6 Liters/min. The BAU can contain steam, and the steam can have atemperature of about 35° C. to about 65° C., about 40° C. to about 60°C., about 45° C. to about 55° C., or about 50° C. The BAU can containsteam that has a flow rate of about 0.1 Liters/min to about 50Liters/min, about 1 Liters/min to about 50 Liters/min, about 10Liters/min to about 50 Liters/min, about 20 Liters/min to about 40Liters/min, about 0.2 Liters/min to about 0.9 Liters/min, about 1.1Liters/min to about 9 Liters/min or about 30 Liters/min.

In order to amplify viral aerosols for suitable detection, the steam maybe warm but not so hot as to inactivate the viral aerosols. For otherliving bioaerosols, it may be necessary to adjust the steam temperatureso as to not kill the organisms. Air with or without bioaerosols in theBAU may also contain non-biological aerosols (sand particles, etc). Ifit is desired to amplify non-biological aerosols, steam temperatureshigher than 65° C. may be used within the system. It would be apparentto one skilled in the art to adjust the steam temperature accordinglydepending on the application and the desired aerosol one wishes toamplify.

In an embodiment, the BADS contains a biosampler. The biosampler cancomprise a collection vessel containing a collection reservoir (andoptionally a collection medium) and can be in fluid connection with thechamber of BAU. Amplified bioaerosols can be drawn into the collectionvessel of the biosampler from the BAU and collected within thecollection reservoir, in the collection medium within (if present). Thebiosampler can contain an impinger to aid in collecting amplifiedbioaerosols for analysis. In an embodiment, amplified bioaerosols can bedrawn into the collection vessel of the biosampler and driven by aninertia-based collection method or device into a collection medium inthe collection reservoir. In an embodiment, the inertia-based collectiondevice can be an electrostatic precipitator. In an embodiment, thecollection reservoir can contain a collection media, and the collectionmedium can be a fluid. In an embodiment, the collection reservoir lacksa fluid collection media. In an embodiment, the collection media is theliquid that is condensed on the bioaerosols during bioaerosolamplification. The fluid of the collection medium can be culture media.The fluid of the collection media can be water or otherwise have theproperties similar to water. The collection media can contain acomponent (for example an enzyme with a lysis function) and/or bufferthat breaks parts of the viral particles down and preserves only certaincomponents of viral particles, such as intact nucleic acids and/orproteins or fragments thereof. In an embodiment, amplified viralaerosols are drawn from the chamber of the BAU into the collectionvessel of the biosampler, where they are driven into the collectionmedium of the collection reservoir by electrostatic precipitation.

In an embodiment, the amplified bioaerosols collected in the biosamplerof the BADS are delivered to a bioaerosol analysis platform (BAP). Thebiosampler can be in fluid connection with the BAP. The amplifiedbioaerosols can be delivered from the biosampler to the BAP with a pump,such as a peristaltic pump. The BAP can detect viral aerosols. The BAPcan contain a bioaerosol detection assay. The BAP can contain amicrofluidic device to analyze bioaerosols and amplified bioaerosols. Inan embodiment, the BAP uses one or more microfluidic devices to analyzeand/or detect bioaerosols or components thereof. In an embodiment, theBAP uses one or more microfluidic devices to analyze amplified viralaerosols. In certain embodiments, the BAP can receive bioaerosolsdelivered manually by the user, by a device such as a micropipette orother suitable pipetting device.

The BAP of the BADS can include one or more bioaerosol detection assays,such as a nucleic acid detection assay or an immunoassay. A bioaerosoldetection assay can detect or analyze bioaerosols and/or componentsthereof. A detection assay could also include a purification method suchas high-pressure liquid chromatography (HPLC), optical detection method,or any other conventionally used detection method to detect bioaerosolsor bioaerosol components such as, but not limited to, proteins, nucleicacids, lipids, and/or carbohydrates. A bioaerosol detection assay canuse a molecular beacon and generate a chemiluminescent or fluorescentsignal in response to the presence of a bioaerosol or componentsthereof. The nucleic acid detection assay can be an isothermalribonucleic acid detection assay, such as nucleic acid sequence-basedamplification (NASBA) or reverse-transcription loop-mediated isothermalamplification (RT-LAMP). The nucleic acid assay can utilize molecularbeacons and detect amplicons by generating a fluorescent or colorimetricsignal. An immunoassay can be configured to detect bioaerosolcomponents, such as nucleic acids, proteins, and/or molecules on virussurfaces. Bioaerosol components can be nucleic acids, proteins, lipids,carbohydrates, or any other constituent that constitutes structureand/or function of the bioaerosol. The immunoassay can be anenzyme-linked immunosorbent assay. The immunoassay can generate acolorimetric signal. The nucleic acid detection assay or immunoassay canbe carried out with the assistance of magnetic beads. The nucleic aciddetection assay can have lysis, wash, and detection steps. The nucleicacid detection assay can be carried out in thin-walled polymerase chainreaction (PCR) tubes or centrifugal tubes. The immunoassay can beperformed with an immiscible phase separation device as described hereinor other formats known in the field. The bioaerosol detection assay canbe performed on or within a microfluidic device, a paper-basedmicrofluidic device, or a laminated paper-based microfluidic device.

A microfluidic device of the BAP within the present BADS can beconstructed from plastic, glass, or paper substrates, or other suitablesubstrates. In an embodiment, the microfluidic device substrate ispaper. A paper-based microfluidic device can be constructed byimpregnating a hydrophobic photoresist into a paper substrate followedby patterning via photolithography to create hydrophobic boundaries. Apaper-based microfluidic device can also be constructed by creatinghydrophobic boundaries in hydrophilic paper by printing a pattern of waxand heating the wax so it is impregnated into the paper substrate.Physical boundaries for fluid flow can be mechanically, chemically, orotherwise etched into the substrate of microfluidic device with asuitable etcher and/or etchant. Physical boundaries for fluid flow on amicrofluidic substrate can also be created with printed plastic orpolymer composition. Hydrophobic and/or physical boundaries are designedto restrict the flow of fluid in a certain direction.

In an embodiment, the microfluidic device substrate is paper. The papercan be a chromatography paper and can be a porous paper and/orhydrophilic paper. The paper-based microfluidic device can be cut from asheet or roll of paper with a cutting device, such as a craft cutter.The sheet or roll of paper can be affixed to a carrier sheet in order toincrease rigidity during cutting. A sacrificial polymer film can beplaced on the paper during cutting/fabrication to reduce tearing. In anembodiment, the microfluidic paper device can optionally be laminatedwith a laminate to improve mechanical strength. In an embodiment, thelaminate is a polyester or other polymer film. The area of the laminatecan be slightly smaller than the area of the paper substrate to allowfor un-laminated areas of paper substrate where reagents or samples foranalysis can be applied. Laminating can be accomplished through a devicesuch as a heated roll laminator or a common clothes iron. The spacingbetween the rollers of a roll laminator can be adjusted to adjust thecompression and effective pore size of the paper and therefore the flowrate of fluids within the device. The microfluidic device of the BAP canbe a paper-based analytical device, a paper-based microfluidic device, alaminated paper-based analytical device (LPAD), or a laminatedpaper-based microfluidic device.

The microfluidic device of the BAP of the present BADS can have a sampleinlet and/or outlet. The sample inlet can receive amplified bioaerosolsamples from the collection medium of the collection reservoir. Thesample inlet can be in fluid communication with the collectionreservoir. The sample inlet can be a small area of un-laminated papersubstrate and can be a sample pad. The sample outlet can pass bioaerosolsamples in collection medium to another microfluidic device. The sampleinlet and sample outlet can be in fluid communication via a channel orother means. In an embodiment, the microfluidic device optionally hasmore than one channel in parallel between the collection reservoir andsample inlet to improve sample throughput. In an embodiment, amplifiedviral aerosols in the collection medium of the collection reservoir aredrawn into a channel of the microfluidic aerosol analysis device throughthe sample inlet. In an embodiment, amplified viral aerosols are drawninto several channels connected in parallel of the microfluidic aerosolanalysis device through the inlet. In an embodiment, the microfluidicdevice and BAP together can comprise a paper-based microfluidic virusplatform (MVP).

A microfluidic device of the BAP of the present BADS can contain asample pad to which a microliter-scale volume of collection mediumcontaining amplified bioaerosols and/or components of amplifiedbioaerosols from the collection reservoir is delivered. The sample padcan be an area of un-laminated paper substrate. Collection medium can bedelivered to the sample pad via a micropipette or a syringe. Collectionmedium can be delivered to the sample pad through an automated fluiddelivery means, such as a tube in fluid communication with thecollection reservoir and a pump that delivers fluid from the collectionreservoir to the sample pad.

The microfluidic device of the BAP of the present BADS can contain oneor more detection zones in fluid communication with the sample padthrough one or more channels in the microfluidic device. In anembodiment, the microfluidic device can contain sample pad and adetection zone for a negative control. In an embodiment, themicrofluidic device can contain a sample pad, a detection zone for anegative control, and one or more detection zones for positive controls.In an embodiment, a positive control can be bovine serum albumin (BSA).In an embodiment, a positive control can be glucose. In an embodiment,the microfluidic device can contain a sample pad, a detection zone forBSA, a detection zone for glucose, a detection zone for a negativecontrol. In an embodiment, the microfluidic device can have a samplepad, a detection zone for a negative control, and one or more aerosoldetection zones. In an embodiment, the microfluidic device can have asample pad, a detection zone for a negative control, and one or morevirus detection zones. The detection zones can detect viruses. Thedetection zones can detect viral components, such as nucleic acids orproteins. If more than one virus detection zone is present, thedetection zones can detect different viruses and/or viral componentsrespectively, or the same virus and/or viral components. If more thanone virus detection zone is present, the detection zones can befunctionally linked to different detection assays. The sample pad anddetection zones can be of any geometric shape. In an embodiment, themicrofluidic device is an LPAD or laminated paper-based microfluidicdevice with an un-laminated sample pad, a detection zone for a negativecontrol, and one or more detection zones for viruses and/or viralcomponents.

The sample pad and/or detection zone[s] of the microfluidic device ofthe BAP can be functionally coupled to one or more bioaerosol detectionmethods or assays. A bioaerosol detection method can be a protein assay.A bioaerosol detection method can be an immunoassay to detect protein.In an embodiment, a detection zone of a paper microfluidic device can becoupled to a protein assay that can include dried citric acid buffer anddried tetrabromophenol blue. The dried citric acid buffer can be of pHabout 1.8. The bioaerosol detection method can be chemiluminescentassay. In an embodiment, the detection zone of a paper microfluidicdevice is coated with chitosan, followed by cross-linking using anamine-reactive bifunctional molecule (for example glutaraldehyde). Thebioaerosol detection method coupled to a detection zone can be animmunoassay. The bioaerosol detection method can be a non-competitive orcompetitive immunoassay, homogenous or heterogeneous. An immunoassay ona detection zone can use a variety of probes, for example peptide ornucleic acid. The probe in an immunoassay can be coupled to anelectrically charged electrode. An immunoassay on a detection zone canemploy one or more reporting methods, including but not limited toenzyme-linked reporting, radio-isotope decay reporting, DNA reporters,flourogenic reporters, electrochemiluminescent reporters, or alabel-less reporter method (such as a surface plasmon resonance ormeasuring change in electrical resistance upon antigen binding to anelectrode). Bioaerosols can be detected from the detection zone of amicrofluidic device using luminol and horseradish peroxidase (HRP). Theimmunoassay coupled to a detection zone can detect protein. Theimmunoassay can be configured to detect viral protein. Detection methodsor assays may need optimization by varying parameters such as reagentconcentration or incubation time, for example, for optimal performance.

In an embodiment, the bioaerosol detection method coupled to a detectionzone of a microfluidic device of the BAP is a nucleic acid detectionmethod. In an embodiment, the nucleic acid detection method coupled to adetection zone is an isothermal amplification reaction for the detectionof ribonucleic acid (RNA), such as nucleic acid sequence-basedamplification (NASBA) or RT-LAMP. In an embodiment, a detection zone iscoupled to an immunoassay that detects viral protein. In an embodiment,a detection zone is coupled to an immunoassay that is configured todetect viral nucleic acid. Multiple microfluidic devices can be in fluidcommunication with one another and functionally coupled within abioaerosol analysis platform in an array to increase detectionthroughput.

The detection assays of the detection zones of the microfluidicdevice[s] of the BAP can generate an amplified bioaerosol detectionsignal. The detection signal can optionally be broadcast through a wired(fiber or GigE for example) or wireless (cellular, bluetooth, and/orWiFi for example) means to a device configured to detect, receive,and/or process the detection signal. The signal can be detected andanalyzed by an optical device, for example a smartphone with lens,imaging sensor, and an application configured to receive and processdata from the imaging sensor, and a display configured to display datafrom the application.

Herein described is a method for detecting bioaerosols. The method cancomprise the steps of: providing an BADS; delivering air containingbioaerosols to the amplified aerosol detection system; adiabaticallycooling the air containing bioaerosols in the bioaerosol amplificationunit of the BADS, wherein the air containing aerosols is cooled within achamber of the bioaerosol amplification unit, wherein the chamber of thebioaerosol amplification unit has one or more interior surfaces adjacentto the air containing bioaerosols, wherein the one or more surfaces arewetted with warm water; mixing cooled air containing aerosols with warmsteam in the chamber of the bioaerosol amplification unit; deliveringair containing amplified bioaerosols from the bioaerosol amplificationunit to a biosampler of the BADS, wherein the biosampler is configuredto receive the air containing amplified bioaerosols through a sampleinlet and collect amplified bioaerosols in a collection reservoir;precipitating, concentrating, or both the amplified bioaerosols into thecollection reservoir of the biosampler; delivering the collectedbioaerosols from the collection reservoir of the biosampler to abioaerosol analysis platform, wherein the bioaerosol analysis platformis functionally coupled to one or more bioaerosol detection assays; anddetecting bioaerosols with one or more detection assays. The bioaerosoldetection assay can be nucleic acid detection assays, such as NASBA orRT-LAMP, or an immunoassay, such as an ELISA, that is configured todetect one or more bioaerosols or components thereof. The detectionassays can further be configured to detect viruses. The bioaerosolamplification unit of the method can be a cylinder with an interiorchamber. The cylinder can have a sample inlet and sample outlet, and thewalls of the chamber can be covered in a wick that is wetted with warmwater. The chamber can also have a drain to drain excess water from thewick. The chamber can be connected to a vacuum pump to draw in aircontaining bioaerosols and can be in fluid communication with a steaminlet, which provides warm steam to the chamber to mix with theadiabatically cooled air containing bioaerosols. It is important thatthe steam not be too hot so that bioaerosols are not inactivated. Therecan also be collection media in the collection reservoir of thebiosampler which amplified bioaerosols are collected into. Thecollection media can contain a bioaerosol stabilization component and/orcan contain a bioaerosol lysis component. The collection media canpreserve components of bioaerosols for detection, such as proteinsand/or nucleic acids. The collection media can be a culture media.

EXAMPLES

Now having described the embodiments of the disclosure, in general, theexamples describe some additional embodiments. While embodiments of thepresent disclosure are described in connection with the example and thecorresponding text and figures, there is no intent to limit embodimentsof the disclosure to these descriptions. On the contrary, the intent isto cover all alternatives, modifications, and equivalents includedwithin the spirit and scope of embodiments of the present disclosure.

Example 1

Viruses are small entities, typically ranging from 20-300 nm, which canreplicate only inside living cells (Prescott et al. 2006). Many virusescan be transmitted through airborne routes, and airborne viruses areresponsible for various diseases in humans, animals and plants, such aschickenpox (by Varicella-zoster Virus, VZV), common cold (bycoronavirus), influenza of humans and animals, rinderpest of cattle andlarge ungulates (by morbillivirus), bovine respiratory disease (byBovine Respiratory Syncytial Virus, BRSV), and some plant viruses thatget aerosolized from soil. The seasonal influenza alone causes3,000-49,000 deaths (Thompson et al. 2003), 3.1 million hospitalizeddays, 31.4 million outpatient visits, direct medical costs of $10.4billion, and lost earnings due to illness and loss of life amounting to$16.3 billion per year in US (Molinari et al. 2007). In 1918, pandemicinfluenza caused 20 million deaths globally. Many deadly viruses havealso been weaponized and intended for offensive use in the form ofaerosols. Hence, sampling and detection of airborne viruses iscritically important for agriculture, animal husbandry, biodefense,conservation, epidemiology, public health, and in general, fordeveloping better protection and prevention strategies for animal andhuman health, public safety and welfare.

Limitations of Current Sampling Techniques for Viral Aerosols

Bioaerosol sampling is typically performed using an impingement method,i.e. directing an air jet containing biological particles to impact onan aqueous collection medium. Because the collection mechanism isinertia based, sampling devices in this category such as All-GlassImpinger (AGI) and the BioSampler® are effective in collectingsupermicron (>1 μm) particles but are less so for particles in thenanometer range. FIG. 1 displays a schematic diagram illustrating thecollection mechanism of an AGI and a BioSampler. Hogan et al. (2005)conducted experiments using bacteriophages MS2 (dia=27.5 nm) and T3(dia=45 nm) to characterize their collection efficiency in the AGI-30,the BioSampler and a frit bubbler. FIG. 2 displays the results. Asshown, the efficiency is terribly low (5-10%) in the 20-100 nm range,which coincides with many viruses. Woo et al. (2012) later also verifiedsuch a trend and developed a “correction factor” for estimating theactual value. However, such a factor is subject to large uncertaintysince the actual efficiency is close to noise level. These studiesclearly show the limit of impingement methods for collecting viralaerosols in the nanometer range.

Other common bioaerosol sampling methods also have their limitations forviral aerosols. Electrostatic collectors have a poor chargingprobability for nanometer particles (Hogan et al. 2004), e.g. 5.2% for20 nm particles. In addition, the production of ozone at high electricalfield intensity can damage viruses (Cox 1987). Filters have highphysical collection efficiency. However, they cause more structuraldamages than other methods. Their extraction is often inefficientbecause nanometer entities adhere strongly on surfaces (Tseng and Li2005; Verreault et al. 2008). An Ultraviolet Aerodynamic Particle Sizer(UV-APS) is a real-time bioaerosol sampling instrument which measuresfluorescence from fluorophores (i.e. nicotinamide adenine dinucleotidephosphate (NADPH) and riboflavin in a live microorganism) excited bypulsed-UV laser to determine viable bioaerosols (Agranovski et al. 2003;Agranovski and Ristovski 2005). Many researchers have considered usingthe UV-APS for viral aerosols, but none has been able to correlate thedata reads with the presence of agent. Furthermore, sensitivity ofdetection by the UV-APS is a big issue for virus aerosols because oftheir nanometer particle size, and lack of fluorophores. Recently, asilicon nanowire sensor functionalized using antibodies was developedfor monitoring airborne virus by utilizing the change in conductance inthe presence of a virus particle (Shen et al. 2011). While successfuldetection of influenza virus was demonstrated, the system is stillplagued by the low charging efficiency of an electrostatic system fornanometer particles. In short, all the above known devices areinefficient for viral aerosol sampling. Hence, the development of adevice capable of efficient viral aerosol sampling is greatly needed.

To overcome the inertia limitation for viral aerosols, one possiblesolution is to amplify nanometer virus particles to much largersupermicron particles, which would then be efficiently collected by aninertia based method. Particle size enlargement by condensation ofalcohol vapor has been realized for non-biological aerosols for decades(Agarwal and Sem 1980). The commercial instrument, i.e. condensationparticle counter (CPC), adopting this principle has one heating chamberfor vaporizing alcohol from an alcohol reservoir to saturate the sampleair, followed by one cooling chamber to create a supersaturationcondition (S=actual vapor pressure/saturation vapor pressure >1) thatwould allow condensation of alcohol on aerosols. As alcohol inactivatesviruses, using an alternative condensing material such as water isdesired for viruses. However, simply replacing alcohol by water usingthe same design as the conventional CPC does not work. This is becausethe higher molecular diffusivity of water vapor compared to the thermaldiffusivity of air coupled with the temperature gradient in thecondensation chamber limit particle growth by causing condensation tooccur predominantly at the colder chamber wall rather than on particles(Hering et al. 2005). This explains why the system built by Milton andcoworkers (McDevitt et al. 2013; Milton et al. 2013) had a performance“comparable” to the BioSamplers when they used water vapor forcollecting infectious influenza aerosols from exhaled breath frompatients. Oh et al. (2010) observed the same problem when they used thecooling principle for collecting 28 nm MS2 viral particles in alaboratory setting, but they successfully improved collection byswitching to a “mixing” chamber (mixing of a cold aerosol flow with ahot moist air flow) to create a proper supersaturation environment thatactivated condensation on particles (Kousaka et al. 1982; Wu et al.2013).

Conventional Methods for Identifying and Quantifying Viruses

Once sampled, several techniques are available for identifying the virusand quantifying the amount. Conventional methods for quantifying virusare plaque assay and 50% tissue culture infectious dose (TCID₅₀), whichrequire culturing of the host cells first followed by infecting thehost. They are important tools for measuring viral infectivity, but arerather time-consuming. Other virus detection methods includeenzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction(PCR). ELISA relies on a specific antibody and a color change toidentify a virus. Briefly, a specific antibody attached to an enzymebinds to its target on the virus. The enzyme reacts with a colorsubstrate resulting in a visible signal. PCR is a biochemical techniquewherein a DNA template is “amplified” through repetitive copying cyclesby a DNA polymerase. Unfortunately, many airborne viruses contain RNAgenomes. If RNA is the template, a preliminary step referred to as“reverse transcription” is necessary to produce a DNA template for PCR.Standard ELISA and PCR do not require viable (infectious) viruses, butthese procedures are still complex and slow, and therefore cannot beused for rapid detection in field investigation. If they can be modifiedto perform like the rapid influenza diagnostic testing based on lateralflow immunochromatographic assays, a form of ELISA, they will bepowerful tools for virus detection in the field.

Microfluidics Technology

Reaction kinetics is controlled by concentration and volume. Hence, byconfining the reagents in a smaller volume, it is then possible toenable faster processing. Microfluidics can be a perfect platform toachieve this goal. Microfluidics technology has been used to constructminiaturized analytical instruments called “Lab-on-a-chip” devices. Theprinciples of microfabrication and microfluidics, as well as theircurrent and potential applications, have been reviewed in the literature(Arora et al. 2010; Whitesides 2006). Common analytical assays,including PCR, protein analysis, DNA separations, and cell manipulationshave been reduced in size and fabricated in a centimeter-scale chip. Thesize reduction of an analytical instrument has many advantages includinghigh speed of analysis, minimization of required sample and reagents,and ability to operate in a high-throughput format. Most microfluidicdevices can be made from silicon, glass, or plastics, as reviewed byManz's group (Arora et al. 2010). A variety of glass, plastic, and paperdevices can be fabricated for various applications including: DNAanalyses (Boone et al. 2002; Fan et al. 1999), protein separation (Dasand Fan 2006; Das et al. 2007; Tan et al. 2002), bacterial and toxindetection (Koh et al. 2003; Mei et al. 2005; Mei et al. 2006), andprotein expression (Khnouf et al. 2009; Mei et al. 2010; Mei et al.2008). Plastics can be used because of (1) biocompatibility of plastics(evidenced by plastic labwares); and (2) processes in manufacturinglow-cost, high-volume plastic parts with micro-scale features (e.g.,compact disc, a two-layer structure containing micron-scale features).The fabrication process of plastic microfluidic devices has beendescribed previously (Boone et al. 2002; Fredrickson et al. 2006). Inaddition, microfluidic devices can be fabricated in and/or on a papersubstrate. From litmus papers to over-the-counter pregnancy test-kits,paper can be used as an analytical platform. Over the past few years,paper has garnered increasing interest as an option for producingmicrofluidic devices. This growing interest in paper-based microfluidicsis driven by several factors. First, paper as a substrate can simplify amicrofluidic system because it is a porous media capable of pumpingaqueous solutions through capillary action. Thus, accessories such as apump may not be needed. Second, paper as an industrial product can beinexpensive, widely available, and derived from renewable resources(Martinez et al. 2007). Paper devices can be fabricated by at least twomethods (Li et al. 2012; Yetisen et al. 2013). The first method canpattern and manipulate the hydrophilic property of the paper substrateby impregnating a hydrophobic photoresist (SU-8) into paper, followed bypatterning via photolithography (Martinez et al. 2007; Martinez et al.2008). An alternative approach to create the hydrophobic boundaries inhydrophilic paper can be to print a pattern of wax, followed by heatingto allow wax to penetrate into paper (Lu et al. 2009; Lu et al. 2010).The second method can be to form physical boundaries by cutting paperusing a laser, knife cutter or plotter to form physical channels (Fentonet al. 2008; Yu et al. 2011). In these devices, either hydrophobicboundaries or physical boundaries can restrict the flow of a fluid in acertain direction.

Paper-based microfluidic devices can be laminated to increase mechanicalstrength and durability of the devices (Cassano and Fan 2013). In a waysimilar to making an identification (ID) card as shown in an embodimentin FIGS. 3(a)-(e), a digital craft cutter can be used to createchromatography paper strips based on the designed pattern. The paperstrips, cover film, and bottom sheet can be aligned and assembledtogether as shown in FIG. 3a . The cover film can have a cutout that isslightly smaller than the paper strip so that the paper strip can beaccessed for reagent dispensing and sample applications. The assemblycan be passed through a heated laminator (FIG. 3b ). As the polyesterfilms are heated, they can conform to the outline of the paper strip(FIG. 3c ). Both cover and bottom films can be made from polyester,providing mechanical backing to the paper device. A picture of anexemplary LPAD is shown in FIGS. 3d and 3e . By encapsulating the paperstrip between layers of thermally bonded polymer films, low-cost andrugged devices can be produced.

Need for a Novel Viral Aerosol Detection System

Because of the poor sampling efficiency and complicated laboratorysystems required for conventional analysis, current knowledge of viralaerosol is rather limited (Xu et al. 2011). For example, how influenzais transmitted is still hotly debated even after decades of research.Thus, a system that can enable highly efficient sampling and fastdetection of viral aerosols will bring great benefits to our society.The present disclosure is directed to a Highly Efficient and RapidBioAerosol Detection System (HERBADS) that can combine particle sizeamplification through condensation of water vapor and rapid analysisthrough microfluidic units. This system can be a suitable system tofulfill the goal and need of rapid, efficient, and precise analysis ofviral aerosols.

The HERBADS is a system that can include a BioAerosol Amplification Unit(BAU) for enhanced sampling efficiency and a paper-based MicrofluidicAerosol Analysis Platform (MAAP) for rapid detection. Each unit can be auseful device itself for improved collection and detection,respectively. Each unit can be combined and integrated. When combined,the integrated system can offer a suitable tool for field investigation.

BioAerosol Amplification Unit (BAU)

The BAU can be based on the principle of water vapor condensation asdescribed in our recent patent (Wu et al. 2013) which has beensuccessfully proven for increasing MS2 bacteriophage capture efficiencyas reported in Oh et al. (2010). To significantly improve itseffectiveness and to compact the size, three new features can beincorporated into the BAU: adiabatic cooling, swirling mixing, and/orwetted walls. FIG. 4 displays an embodiment of the system. Thebioaerosol stream, which can be a viral aerosol stream, can first beadiabatically cooled to start the initial condensation. The particlesize amplification can further be accelerated by swirling mixing withwarm steam. The wetted wall can supply more water vapor and can preventaerosol deposit onto the chamber wall. The amplified aerosols can thenbe efficiently collected by a collection apparatus, for example aninertia-based apparatus downstream such as a BioSampler. The details ofeach feature are further discussed in the following subsections.

Adiabatic Cooling

Supersaturation can be a necessary condition for suitable condensation.Herein, adiabatic cooling can be a suitable method to createsupersaturation. When an air stream expands adiabatically, itstemperature decreases (FIG. 5a ) according to the following relationshipin equation 1 (Friedlander 2000):

$\begin{matrix}{\frac{p_{final}}{p_{inital}} = \left( \frac{T_{final}}{T_{initial}} \right)^{{\gamma/\gamma} - 1}} & {{Eq}.\mspace{11mu} 1}\end{matrix}$where p and Tare pressure and temperature, γ is the ratio of specificheats (1.4 for dry air and 1.33 for water vapor). Since saturation vaporpressure decreases with temperature decrease, air becomes more saturatedand condensation is initiated when saturation ratio (S) exceeds 1 (FIG.5a ). FIG. 5b conceptually illustrates the process in an expansionnozzle to be adopted for the new design. In the BAU, the bioaerosols inthe supersaturated stream can act as the nuclei for water vaporcondensation that yields size amplification. This process is similar tocloud formation in the atmosphere. As shown in Eq. (1), the temperaturedrop is controlled by the pressure ratio (after vs. before expansion),which is an operating parameter in the BAU.Swirling Mixing

Close contact between the aerosol and the condensing vapor can be ofseminal importance in the condensation process and the degree ofcloseness can dictate the particle growth rate. Conventional wisdomrelying on molecular diffusion in a laminar flow can work, but thegrowth rate is constrained by the molecular diffusion rate. In the BAU,a swirling mixing process, for example as proposed by Buesser andPratsinis (2011)(for coating a silica layer onto host titania particlesin their work), can be implemented in order to significantly enhance themixing in the BAU. The cold aerosol stream from the adiabatic coolingcan enter the chamber from the center, while warm steam (not overly hotto avoid virus deactivation) can enter from the side with an angle (seeFIG. 4(a)). Such a configuration can induce a swirling aerosol motion inthe mixing zone. FIGS. 6(a)-(b) displays the flow streamlines in anexample configuration. The mixing ratio (warm steam flow rate/coldaerosol flow rate), as inferred, can be a key operating parameter. At alow mixing ratio (FIG. 6a ) the exchange between the aerosol flow andthe steam can be rather slow. In contrast, a high mixing ratio (FIG. 6b) can yield improved mixing through the swirling action. In other words,the water vapor can better reach the reactor center and this can lead tobetter exposure of all core aerosol streamlines to the steam. Oneadditional advantage of such a configuration is its high temperaturegradient between the warm steam and cold aerosol that can further drivethe condensation of water vapor onto the cold aerosol surface. Byimproving the effectiveness of mixing, the length of the mixing chambercan be reduced, thus enabling a more compact design.

Wetted Wall

A cold surface can induce water vapor condensation due to a temperaturegradient between the warm steam and the cold wall, thus depleting theavailable vapor needed for amplifying the aerosol size. This can be areason why the cooling chamber design does not work for watercondensation (Hering et al. 2005). The temperature gradient can alsoundesirably drive the amplified aerosols depositing onto the wall. Awetted wall saturated with warm water can overcome this barrier. Byproviding a higher surface temperature, a “reverse” temperature gradientcan be created that “pushes” away approaching aerosols. Furthermore, thewarm water released from the wall can replenish the vapor supply, whichcan further enlarge the aerosol size. The wetted surface can be createdby using a porous and hydrophilic wick (FIG. 4(a)). It can maintainconstant wetting by means of a small pump that injects water into thewick at the upper end of the growth chamber. Water can spread bycapillary action and by gravity down the wick, and the excess can drainout the chamber at the bottom (FIG. 4(a)). To ensure the drain does notbecome a safety concern, a filter can optionally be used to captureviruses running off in the drain. Nonetheless, the temperature gradientof the wetted wall design can be suitable to minimize such a need.

Incorporating and integrating all the above designs into the system canbe complex. To efficiently determine the optimal configuration andoperating conditions, computational fluid dynamic simulation coupledwith aerosol dynamic modeling can be conducted. The modeling can becarried out, for example, using FLUENT (ANSYS) for fluid dynamicscoupled with Fine Particle Model (FPM by Chimera) for aerosol dynamicsin parallel on a workstation. FLUENT is a widely used software packagethat uses numerical methods and algorithms to solve and analyze theinteraction of fluid with surface defined by boundary conditions forfluid, heat transfer, and reaction. FPM is designed to model aerosoldynamics including particle formation, growth and transport. The FPMuser interface is tightly integrated with the ANSYS FLUENT thus allowingeasy setup of standard particledynamics simulations. First the flow ateach time step is determined by FLUENT; the data can then be used asinput in the FPM to determine the interaction between aerosols and watervapor. Sensitivity analyses can be carried out for various importantoperating parameters, including pressure ratio across the nozzle, mixingratio of the warm stream to aerosol flow, temperature of the wettedsurface. A BAU can then be built according to the optimal configurationdetermined by the modeling.

Amplified Bioaerosol Detection Platform and Paper-Based MicrofluidicDevices for Bioaerosol Detection

Device Design

Amplified bioaerosols can be detected by an bioaerosol analysis platform(BAP). Microfluidic devices can be used within a BAP for bioaerosoldetection, such as viral detection, as shown in FIGS. 7(a) and (b).These layouts show the versatility of the platform, and differentdesigns can be adapted if different assay and detection schemes arerequired for the intended use. FIG. 7a shows colorimetric assays forsimultaneous detection of two viruses. The microfluidic device caninclude a sample pad, detection zones for virus #1 and virus #2, and anegative control. Reagents can be applied to the detection zone afterlamination. For instance, the detection zone can be deposited withcitric acid buffer at pH 1.8, followed by drying. It is then spottedwith tetrabromophenol blue and allowed to dry again. When a virus sampleflows into the detection zone, protein on virus surfaces would make thedetection area to turn brownish color (if there are enough proteins).

FIG. 7b shows an alternative embodiment of a microfluidic device designbased on chemiluminescence detection. Among many detection methods,chemiluminescence can be one of the more sensitive methods due primarilyto little or no background signal. As a result, chemiluminescence can beused for bioaerosol detection, especially for viral aerosol detection.The protocol for implementing the assay can be as follows. First, thedetection zone of a paper device can be coated with chitosan, followedby cross-linking using an amine-reactive bifunctional molecule(glutaraldehyde) in a way similar to those reported in the literature(Wang et al. 2012). Chitosan adheres to paper due to electrostaticinteractions between positively charged chitosan and negatively chargedcellulose in the wet condition. Glutaraldehyde is immobilized onto thesurface through amino groups of chitosan. It can also bind covalentlywith a virus antibody (anti-virus).

The embodiment illustrated in FIG. 7b is a competitive immunoassay incase a tiny virus does not have multiple epitopes for both captureantibody and detection antibody. Otherwise sandwich immunoassay formatcan be easily adapted. For competitive immunoassays, a sample solutioncontaining virus and a known amount of horseradish peroxidase (HRP)conjugated virus (virus-HRP) can be applied to the detection zone. Bothsample virus and virus-HRP can compete with each other for the fixedamount of anti-virus immobilized in the detection zone. After washing, asolution of luminol and hydrogen peroxide can be dispensed to thereagent pad, and flow into the detection zone. The resultantchemiluminescent signal can then be detected, and it can correlate withthe amount of the viruses in the sample.

Array Detection

The bioaerosol in FIG. 7b can be easily replaced with proteins expressedby a bioaerosol particle, such as a virus, or other biomarkers relatedto bioaerosols, such as hemagglutinins and neuraminidase of influenzaviruses for example. An array of microfluidic devices, such aspaper-based microfluidic devices, can be made in the same amplifiedbioaerosol detection platform so that a pattern can be generated toaddress the possible cross-reactivity of antibodies for a range ofbioaerosols, such as influenza viruses. The fluidic format of a proteinarray reported in the literature can be possible for profiling varioustypes of bioaerosols, such as the influenza virus (Koopmans et al.2012). Array-based detection in this sense can be more accurate thancommercially available lateral flow assay kits for bioaerosols, such asthe influenza virus or other viruses or bioaerosols that are based onone protein. The pattern-recognition from an array using a number ofbiomarkers can reduce or eliminate false-positives and false-negativesthat are often encountered from an assay using a single biomarker.

Low Sample Volume

One common concern about microfluidics-based method is the samplevolume. Sometimes sample volume or collection media can be low, andthere can be a potential issue regarding the sampling accuracy. Forexample, when the virus concentration in a sample solution is 10 virusparticles/mL, processing 100 μL of the sample solution should detect 1virus particle on average, but with a possibility of zero virusdetected. As a result, at least 300 to 500 μL of the sample solutionshould be processed to ensure accurate sampling. If sample bioaerosolvolume in the present system is low, device throughput can be increasedin the amplified bioaerosol detection platform with the addition ofparallel microfluidic channels. FIG. 8 shows the picture of anembodiment of one glass device including 8 channels that are connectedthrough bifurcations. A device such as this can process 2 μL/s bloodsamples for example, and can be designed to capture rare circulatingtumor cells in peripheral blood (Sheng et al. 2012). Typically thenumber of tumor cells in cancer patients can be about 10 tumor cells/mL,thus 1 mL of blood must be processed to ensure sampling accuracy. Inthis device, one mL of blood takes 8.3 minutes to pass. A device such asthis can be incorporated into the presently described BADS to improvebioaerosol throughput and ensure accurate detection and sampling of lowconcentrations of bioaerosols. Devices can be constructed incorporatingparallel channels out of a thermoplastic substrate and incorporated intothe presently described system. Both colorimetric and immunoassaydetections discussed in FIGS. 7(a)-(b) can be incorporated into thedevice and system as a whole.

System Integration

As amplified bioaerosols, such as viral particles, can be efficientlycollected by inertia based methods, impingement devices such as aBioSampler can be a used to collect the viruses and serve as areservoir. A sample can then be easily retrieved and delivered to theamplified bioaerosol detection platform by a simple peristaltic pumpwith tubing connecting the reservoir and the amplified bioaerosoldetection platform. Hence, one embodiment of integration will be todemonstrate the capability of this BAU-BioSampler-BAP design.

However, for cases where virus concentration is low, the required volumeof collection media in the reservoir (20 mL for the BioSampler) canpossibly dilute the concentration down to below the detection limit ofthe BAP. Hence, an alternative delivery method such as an electrostaticprecipitation (ESP) method can be used (Cheng et al. 1981; Hogan et al.2004) to accomplish the goal. While the charging efficiency fornanosized particles can be low as discussed earlier, supermicronparticles such as the amplified particles can be charged veryefficiently (˜100%). Furthermore, the water content of the amplifiedparticles can shield the viruses from the damaging effect of ozone, abyproduct of corona charging well known to be deleterious to nakedviruses. Thus, electrostatic collection can serve as a useful tool. Insome instances, viral aerosol concentration can be sufficiently high andaerosols sufficiently amplified that impingers or other collectionmethods may not be required. In cases such as this, collection cansimply rely on gravity or other passive method for amplified aerosols tobe collected in the collection media.

FIG. 9 conceptually illustrates an embodiment of a design of a BADS asdescribed herein. Drawn by a vacuum pump, amplified bioaerosols from theBAU will be directed to an ESP unit where the needle electrode generatescorona (crowd of electrons) to charge the amplified bioaerosolparticles. Amplified bioaerosols in this embodiment can be viralaerosols. The charged amplified particles can be subsequently attractedby the downward electrical field to the collection vessel at the bottom.The curve of the vessel can allow the collected particles to settle atthe bottom. Since this method requires no liquid collection medium likethe BioSampler does and only the amplified particles are collected, thevirus concentration in the collection should be higher (i.e. moreconcentrated) than the impingement method. Once sufficient volume ofliquid sample is collected, a peristaltic pump will deliver a minutesample to the BAP. The presence of target bioaerosol, such as a virus,can then be detected statically or as a function of time as controlledby the sampling rate.

Performance Testing

Individual Unit Testing

BAU embodiments can be tested and their performance measured usingpolystyrene latex (PSL) particles of known sizes (30, 50, 100 and 300 nmcovering the common size range of viruses). As PSL particles come with auniform size, the amplification effect can be examined by monitoring theaerosol size and concentration. Another important reason for such a testusing non-biological particles is that it can require much less timethan testing involving assaying, and can provide a quick but accuratephysical characterization.

An embodiment of an experimental system for testing is shown in FIG. 10a. PSL aerosols can be produced by nebulizing a PSL particle suspension(Oh et al. 2010). The nebulized particles can first be conditioned bydry dilution air to vaporize the water content. The aerosols can bemonitored for size and concentration by a Scanning Mobility ParticleSizer (SMPS) for the nanometer to lower submicron range (<0.5 μm) and byan Aerodynamic Particle Sizer (APS) for the upper submicron tosupermicron range (>0.5 μm). The aerosols going through the system withthe BAU off (i.e. no adiabatic cooling, steam and wetted wall) can serveas the baseline for comparison. The comparison of the particle sizes andconcentrations with the BAU on and off can reveal the effectiveness ofthe BAU in amplifying aerosol size.

BAU performance can also be tested with other bioaerosols, such as viralaerosols. MS2 bacteriophage (ATCC 15597-61) can be a candidate virus forthis phase because MS2 only replicates in male E. coli bacteria and issafe to work with in a biosafety level 1 (BSL-1) laboratory. With adiameter of approximately 28 nm, MS2 is a single-stranded RNAicosahedral virus that is commonly used in viral aerosol testing(Grinshpun et al. 2010; Rengasamy et al. 2010; Tseng and Li 2005)because of its similarity to human enterovirus and picornavirus(Aranha-Creado and Brandwein 1999).

The experimental system can be similar to that used for the PSLparticles, except that the aerosols can also be collected by aBioSampler downstream (FIG. 10a ). The collected samples in theBioSampler can be subjected to standard plaque assay[s] (Adams 1959)using E. coli as host bacterium for infectious count as well as reversetranscript-polymeric chain reaction (RT-PCR) to determine the totalvirus count. Again, the comparison of the virus counts (plaque assay andRT-PCR) with the BAU on and off can measure the performance of the BAU.In addition, the liquid drained from BAU can be collected and sampled toexamine if bioaerosols such as viruses escape through the drain, and theloss through the drain can be quantified. If escape is confirmed, afilter can be installed to increase safety of the device.

Regarding the testing of the BAP, different detection methods such ascolorimetric and chemiluminescence detection can be used as discussedabove. Known amounts of viruses can be fed to the BAP unit through asyringe or other suitable method, such as a micropipette. Thecolorimetric method can be simple to implement without a need of aninstrument, while chemiluminescence methods can require a photon counteror photomultiplier tube to measure the light generated. For the former,a colorimetric chemistry similar to those in Quidel QuickVue InfluenzaA+B Test (Quidel, San Diego, Calif., USA) can be implemented inpaper-based analytical devices. Chemiluminescence detection can becarried out as illustrated in FIG. 7b and discussed in the related textabove. Different titers of virus can be used to establish a calibrationcurve and determine the limit of detection.

System Testing

Information learned by using MS2 bacteriophage cannot be used togeneralize about the bioaerosol amplification and detection devicedescribed herein for collection efficacy for virus aerosols becauseviruses have different physical, chemical, and biological properties.For example, MS2 bacteriophages are not covered by a lipid membrane, andare uniform in size, whereas influenza viruses are larger viruses thatare pleomorphic, meaning they are non-uniform in shape and can occur inspherical to filamentous forms. Moreover, influenza viruses are covered(“enveloped”) by a lipid membrane. The stability of enveloped andnon-enveloped viruses in aerosols can be affected by their biochemicalmakeup, and by temperature and relative humidity. In general,lipid-containing viruses are usually more stable in aerosols thanlipid-free viruses, but less stable in moist air than in dry air (Akers1973).

From on-going work (Fennelly et al. 2011), ultrafine aerosols (massmedian aerodynamic diameter ˜0.8 μm) of wild-type influenza virusA/Mexico/4108/2009 (H1N1), the H1N1 strain of the 2009 influenzapandemic, can be generated with minimal loss of viral infectivity usinga Collison nebulizer. This can be a candidate to be used in devicetesting methodological verification. Viral stocks can be prepared inserum-free Madin-Darby canine kidney (MDCK) epithelial cells in thepresence of trypsin using a low-passage virus isolate (obtained from theCDC). A process that conserves the genotype and produces relatively fewdefective particles (WHO 2002) can be used for virus production. Virusidentity can be confirmed by PCR and sequencing before device testing.

System testing with influenza virus can be performed in a USDA inspectedand approved BSL2-enhanced laboratory using a testing system similar tothat illustrated in FIG. 10b . Since influenza virus aerosols in fieldconditions typically are in low concentration, emphasis can be placed ondetecting low-concentration virus aerosols, i.e. using the BAU-ESP-BAPdesign. Influenza virus aerosols can be produced by nebulizing the virusstock, and a targeted volume of air can be sampled. Knowing the titerand liquid volume nebulized, the quantity of influenza viruses withinthe volume of sampled air, N_(sapledAir), can be determined. Thecollection efficiency of the bioaerosol amplification and detectiondevice, or highly efficient and rapid BioAerosol detection system(HERBADS) as used in this example, for influenza virus aerosols over aconcentration range can then be evaluated according to the followingEquation 2:

$\begin{matrix}{{\eta(\%)} = {\frac{N_{HERBADS}}{N_{SampledAir}} \times 100}} & {{Equation}\mspace{14mu} 2}\end{matrix}$where N_(HERBADS) is the quantity of influenza viruses collected by theHERBADS embodiment in the sampled air. Eq. 2 can be used to evaluatecollection efficiency of other bioaerosol amplification detection systemembodiments and methods. Viral infectivity can be determined using aTCID50 assay (Hamilton et al. 2011; Lednicky et al. 2010), and theReed-Muench method can be used to calculate TCID50 values (Reed andMuench 1938). A comparison can be made between viral aerosols throughthe HERBADS with BAU on and off. The corresponding thresholdconcentration for detecting influenza viruses by the BAP will bedetermined.

In parallel, the BAU can also be connected to a BioSampler for testing,which can serve as a baseline for investigating the performance of theelectrostatic method. The liquid sampling medium can includephosphate-buffered saline containing 0.5% purified bovine serum albuminfraction V, which can be suitable for the collection of influenza virusaerosols (Lednicky et al. 2010). Ongoing work (Fennelly et al. 2011) andprevious projects (Anwar et al. 2010), show that the Biosampler can beoperated at a flow rate lower than the manufacturer's recommendedsampling rate of 12.5 L/min. Lower flow rates can improve performancefor original viruses (i.e. non-amplified). Therefore, the BioSampler canbe operated at different flow rates for testing purposes (12.5 and 8L/min, for example) to explore how different collection characteristicsof embodiments of the herein described system[s]. For completion, aquantitative PCR assay can also be used to evaluate the total number ofvirus particles captured by the bioaerosol amplification and detectiondevice, which can verify whether the steam condensation processinactivates influenza virus particles.

FIG. 11 shows particle size distribution of MS2 aerosol with and withoutadiabatic expansion as can be obtained by a BAU described herein. MS2 isa bacteriophage (virus that infects bacteria) that is commonly used as asurrogate for pathogenic viruses. The measurements of aerosol sizedistribution were made by a device that has a lower detection limit ofaround 0.3 μm. As shown, without adiabatic expansion, only a smallfraction of the particles is larger than 1 μm. With adiabatic expansion,many particles are physically amplified to larger than 1 μm that can becollected efficiently.

FIG. 12 shows viable MS2 concentration without adiabatic expansion vs.with adiabatic expansion as can be obtained with a BAU and collectionreservoir of the present disclosure. As shown, with amplificationthrough the adiabatic expansion, more viable MS2 viruses are collecteddue to the higher efficiency for larger particles without losing theirviability. FIGS. 11 and 12 together demonstrate the system describedhere within can suitably amplify bioaerosol particles, specificallyviral aerosols, and subsequently can allow for more efficientcollection.

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Example 2

After the viruses are collected in the collector, one way to detect themis based on their unique nucleic acid sequences. An amplification methodis typically used in combination with a molecular beacon. The purpose ofthe amplification in this example is to increase the copy number ofnucleic acids contained in the collected viruses and then allow them tobe detected reliably above a threshold or noise floor. The amplificationmethods can include polymerase chain reactions (PCR) and variationsthereof, a number of linear amplification methods, and other signalamplification methods. Several examples are discussed below.

In-Tube NASBA with Molecular Beacon

This detection scheme can detect the fluorescence signal of molecularbeacons generated from NASBA amplicons. An exemplary scheme is shown inFIG. 17. After the virus particles are collected in the collector tube,a concentrated virus lysis solution can be added along with magneticsilica beads. The beads with an appropriate coating can capture nucleicacid[s] (e.g., RNA from MS2 or flu viruses) released by the viral lysisstep. Then, a device such as a magnetic holder can be used to attractthe beads to the bottom or the side of the collection tube, which canallow the supernatant to be removed and allow for subsequent simple washsteps and/or reagent addition[s]. With the same method, required washingsteps can be performed before the NASBA reaction mix and molecularbeacons being added to the tube. NASBA can then be performed by heatingthe closed collection tube. If the target virus is present, themolecular beacons can generate fluorescence signal which could bedetected and analyzed by an optical device, for example a smartphonewith lens, imaging sensor, and an application configured to receive andprocess data from the imaging sensor, and a display configured todisplay data from the application.

Amplification of Virus RNA with NASBA

Nucleic acid sequence based amplification (NASBA) can been used toamplify RNA extracted from viruses, such as MS2 and influenza viruses.RNA from MS2 and flu viruses can be extracted using a commerciallyavailable kit, for example the QIAamp® RNA mini kit (QUIAGEN). The NASBAreaction mix cab be prepared in house with reagents from a commercialvendor such as Fisher Scientific®. The primers involved can be designedbased on sequences of interest found in databases, such as NCBI(GenBank: NC_001417.2 can be used for MS2, GenBank: AY139081.1 can beused for flu), and reaction can be carried out at 42° C. for 2 hours.The resulting amplicon of interest can be confirmed by electrophoresisusing an Agilent 2100 Bioanalyzer Instrument. The results of a typicalNASBA reaction showing MS2 and flu viral amplicons are shown in FIGS.15(a)-(d).

Example 3

In-Tube Amplification of Flu Virus RNA with Colorimetric ReverseTranscription Loop-Mediated Isothermal Amplification (RT-LAMP)

Reverse transcription loop-meditated isothermal amplification (RT-LAMP)can be used to amplify flu virus RNA. Colorimetric detection can beachieved by using SYBR Green I under UV light and phenol red underambient light. The preparation of flu virus RNA can be done as describedin the section above. RT-LAMP mixture can be prepared in house withcommercially available reagents from vendors such as Fisher Scientific®and New England BioLabs®. The RT-LAMP reaction can be carried out at63.5° C. for 1 hour. The amplicon can be confirmed both by eye andagarose gel electrophoresis. An example of the result of flu virusRT-LAMP is shown in FIGS. 16(a)-(b). With RT-LAMP method, about 10TCID₅₀ flu virus RNA copies can be detected in a 25 μL PCR tube.

Example 4

ELISA-Based Detection with an Immiscible Phase Separation Device

Another example of a detection scheme is based on immunoassays likeenzyme-linked immunosorbent assay (ELISA). To perform ELISA in apoint-of-care format, an immiscible phase separation device as shown inFIGS. 18(a)-(b) can be used. As shown in FIG. 18(a) this device can haveconnected aqueous phase chambers separated by oil plugs. The oil plugswould prevent the aqueous phases from mixing with each other whileallowing magnetic beads to pass by (guided by moving a magnet along). Toperform the detection, magnet beads conjugated with antibodies can beloaded into the collection tube, and then transferred into the firstchamber of the device as described in the previous section. After thesemagnetic beads interact with target viruses, a user can simply pull themagnet placed beneath the device to move the beads through the oil phaseto next chamber. Different chambers for washing and other steps arerequired as needed and shown in FIG. 18b . If the target viruses arepresent in the collection tube, a colorimetric, fluorescent, orluminescent signal would be generated at the last chamber of the device,as practiced in the last step in traditional ELISA. The immunoassay inthis example can be configured to detect viral components, such asproteins and/or nucleic acid[s].

Example 5

Colorimetric RT-LAMP on Paper-Based Lamination Disc Device

This detection scheme is based on colorimetric RT-LAMP mentionedpreviously. In this scheme, an LPAD is described which can be designedand configured to perform virus lysis, RNA extraction, and RT-LAMPdetection all together in one device. As shown in the example in FIGS.19(a)-(f), this device can include four layers, all of which can befabricated with heat lamination with thermoplastic film and PDMScoating. The first layer can include a sample loading port. The secondlayer can include an RNA extraction unit made of a paper that cancapture and store nucleic acids, such as a commercially availableWhatman® FTA card, chromatography paper, or glass-fiber-based paper. Thethird layer can include multiple cellulose paper discs and can providethe capillary forces necessary for washing. The fourth layer can includea small heater. These four layers can be integrated together with adevice, such as a screw through the hole in the middle of each layer, orother means. To perform the detection, a user can rotate the layers tothe place shown in FIG. 20(a). Then, the sample can be loaded throughthe loading port to the FTA card, allowing the sample to dry at roomtemperature or standard conditions. In the second step, the user canrotate the layers to the place shown in FIG. 20(b). Then, the washingbuffer could be added, and the wash step can be performed by thecapillary forces provided by the paper disc in the third layer. Thethird step can comprise adding RT-LAMP buffer or an amplification bufferin a way similar to the second step as shown in FIG. 20(c). In the finalstep, the layers can be rotated as shown in FIG. 20(d), and then theheater can be turned on to induce DNA amplification. If the target virusis present in the collection solution, RT-LAMP colorimetric mix canchange color after the final step as shown in FIG. 16(b).

Example 6

LPAD Virus Detection Device Design and Fabrication

An LPAD virus detection device can comprise a laminated paper-based RNApurification pad, a double-sided adhesive layer, and a polycarbonate(PC) holder, as shown in FIG. 21(a). FIGS. 21(a)-(b) show an embodimentof an LPAD device which can be used for virus detection, nucleicacid-based virus detection for example. FIG. 21(a) shows an embodimentof construction of a device and FIG. 21(b) shows a photograph of adevice. The laminated paper-based RNA purification pad can be made bylaminating a punch of FTA™ card (Whatman) or other paper between twolayers of thermo-lamination film. The whole device can be assembled byaligning the holes shaped within the double-sided adhesive layer and thePC holder with the FTA™ punch, and pressed together to bond (FIG.21(B)).

Testing the Detection Limit Using H1N1 Flu Virus

To operate the device, in an embodiment, virus-containing liquid samplecan first be mixed with lysis buffer and ethanol to obtain RNA fromvirus capsids, then loaded to the laminated paper-based RNA purificationpad (LPAD) through the device center hole. A paper pad can be pressedagainst the bottom of the device to provide capillary force to filterthe sample solution through the FTA™ punch. Meanwhile, the RNA in thesample can be immobilized onto the FTA™ punch, and then purified bywashing with wash buffers in the same manner. The purified RNA was driedfor 20 minutes in room temperature before RT-LAMP. After drying, thebottom of the device was sealed with a piece of transparent tape orPCR-tape. RT-LAMP buffer was then added into the device, followed bysealing the top of the device with another piece of transparent tape orPCR-tape. Incubation was done by a 40-minute-65° C.-water bath, amicroheater, or other means. The amplicons could then be verified witheither colorimetric methods, traditional gel electrophoresis, or otheramplicon detection methods.

FIGS. 22(a)-(b) show an embodiment of detection of H1N1 flu virus RNAusing an embodiment of the device verified by agarose gelelectrophoresis stained with ethidium bromide. The left lane of eachimage is a 100 bp DNA ladder. The TCID50 number of flu viruses put in adevice is marked above each lane, with the “−” marks the negativecontrol. The detection limit was obtained by testing with 100, 50, 25,10, 5, 2.5, and 0 (as negative controls) TCID₅₀ H1N1 flu viruses in eachdevice. As shown in FIGS. 22(a)-(b), gel electrophoresis and ethidiumbromide staining confirmed that this device could detect down to 5TCID₅₀ H1N1 flu viruses.

Demonstration of in-Device Colorimetric Detection

Both pH-sensitive dye (using phenol red) and DNA-intercalating dye(using SYBR Green I) were tested to demonstrate colorimetric detection.In pH-sensitive dye based method, a buffer-free RT-LAMP solution wasprepared in house to allow pH changes during nucleic acid amplification.FIGS. 23(a)-(d) is a demonstration of an embodiment of in-devicecolorimetric H1N1 flu virus detection using phenol red and SYBR Green Idye. “+” marks a positive sample while “−” marks the negative control.FIG. 23(a) shows embodiments of two devices with phenol red RT-LAMPbuffer before incubation. FIG. 23(b) shows the two devices afterincubation, positive sample device turns to orange from pink. FIG. 23(c)shows RT-LAMP buffer incubated in the device observed under ambientlight after adding SYBR Green I dye. The positive sample shows a lightyellow color while the negative sample shows a darker yellow color. FIG.23(d) show the two samples observed under a blue LED flashlight. Thepositive sample (left) has a green fluorescence.

As shown in FIG. 3(a), the RT-LAMP solution had a pinkish color from thepre-added phenol red before incubation. After incubation, the RT-LAMPsolution from the positive device (containing the target virus) turnedyellow as the pH decreased, while the negative control remained pink(FIG. 3(b)).

In DNA-intercalating dye method, SYBR Green I dye was addedpost-incubation. The RT-LAMP solutions between the positive device andnegative control have a slight color difference observed under ambientlight (FIG. 3(c)). Using a common blue LED flashlight, a strong greenfluorescence was observed in the positive device (FIG. 3(d)),guaranteeing effortless result reading.

Example 7

An Efficient Virus Sampler Enabled by Adiabatic Expansion

Adiabatic expansion, includes an instant volume expansion whereby thereis no heat transfer between the contained volume and its surroundings(Bailyn, 1994). For a system with a certain volume, the temperature,volume and pressure of the system before and after theadiabatic-expansion are related as follows:P ₀ V ₀ ^(γ) =P _(f) V _(f) ^(γ)  (1)T ₀ V ₀ ^(γ-1) =T _(f) V _(f) ^(γ-1)  (2)where P, V, T are pressure, volume and temperature, respectively,subscripts 0 and f refer to before and after adiabatic expansion,respectively, and γ is the ratio of specific heat of relevant gas at aconstant pressure over that at a constant volume (Strey et al., 1986).Aitken (1888) implemented this principle to create supersaturation bylowering temperature of the surrounding air of target dust aerosol.Pollak and O'Connor (1955) applied this principle in their photoelectriccondensation nucleus counter (CNC), wherein a photoelectric sensor wasused to count the number of enlarged mist particles. Pollak and Metnieks(1960) investigated the performance of the CNC under different volumeexpansion ratios

$\left( {{i.e.},\frac{p_{f}}{p_{0}}} \right)$and maturation ratio of 3.50 under a compression ratio of 1.21, whichexceeded the required Kelvin ratio for ultrafine particles (Miller &Bodhaine, 1982b). In their research, the CNC successfully amplifiedparticles as small as 20 nm (Miller & Bodhaine, 1982a). Compared to themixing and cooling approaches, the adiabatic expansion approach canresult in an extremely high supersaturation ratio instantly, whichactivates the growth of particles in a very short time. This highsupersaturation ratio played a key role in activating amplification ofultrafine particles as small as 13 nm (Liu et al., 1984). While therehave been a handful of studies on size enlargement of particles usingadiabatic expansion as discussed, there is no study regarding sizeenlargement of virus aerosol by this approach yet.

The present example was embarked to apply the adiabatic expansionprinciple to engineer a highly efficient size amplification device toaddress limitations associated with previously mentioned methods. Onthis ground, a prototype of Batch Adiabatic-expansion for SizeIntensification by Condensation (BASIC) sampler was designed andfabricated. The BASIC sampler described herein is an embodiment of aBADS as described previously. Performance of the BASIC in regards tosize amplification was evaluated. Since collection of viable virusaerosol was a major purpose for the new device, experiments wereconducted to evaluate its ability in collecting viable viruses. Tooptimize the BASIC's operation, sensitivity analyses on key parameterswere conducted, including compression pressure, number ofcompression/expansion cycles (C/E cycles), temperature of the condensingwater, and dwell time after the expansion.

Materials and Methods

Design of the Basic

The BASIC consisted of an expansion bag in a chamber. The bag containedthe aerosol sample while the chamber was used for providing a room forexertion of compression to and subsequent expansion of the bag (seeFIGS. 24(a)-24(c)). Certain amount of de-ionized (DI) water was placedinside the expansion bag as the source for volatile vapor for latercondensation on the aerosol sample trapped inside the bag. The DI wateralso served as the medium for collecting the amplified viruses. TheBASIC device can be about 21 inches high and about 4 inches in diameter.

Experimental Setup

A schematic diagram of the experimental set-up is shown in FIG. 24(d).First, compressed air was directed to a 6-jet Collison nebulizer (ModelCN25; BGI Inc., Waltham, Mass., USA) at a controlled flow rate of 6 Lpmto generate the aerosol flow. A diffusion dryer was positioned at theoutlet of the nebulizer to remove the water content of the aerosol.Before the aerosol was directed into the BASIC, the expansion bag wasdeflated by filling the chamber with compressed air. Then, the sampleaerosol flow was directed into the expansion bag from the aerosol inletport to fill the bag (FIG. 24(a)) while the air discharge valve was opento release the air in the chamber. Once the bag was fully filled,aerosol introduction was stopped and compressed air was fed to thechamber instead to increase the chamber pressure. After reaching thetargeted compression pressure, the air discharge valve was openedrapidly to swiftly drop the chamber's pressure down to the atmosphericpressure level. Thus, the bag expanded instantly, and supersaturationcondition was realized inside the expansion bag. This process enabledthe particles contained inside the bag to be amplified by watercondensation as temperature rapidly decreased due to the swift pressuredecrease.

Experimental Procedure

Physical Size Amplification

Performance evaluation of the BASIC was split into two phases. In Phase1, the physical size amplification was investigated. Amplified aerosolinside the expansion bag was discharged and directed to an OpticalParticle Counter (OPC Model 1.108, Grimm® Technologies Inc.,Douglasville, Ga., USA; size range 0.3-20 μm) for measurements of totalnumber concentration and count median diameter (CMD) of the supermicronparticles (i.e., d_(p)≥1 μm). The CMD was determined using alog-probability plot of the measured size distribution for locating thecorresponding diameter of 50% cut-off point of the accumulative numberconcentration (Hinds, 1999). Control groups were also included by simplyintroducing the same sample aerosol into the expansion bag withoutapplication of any C/E cycle.

Viability Preservation

Due to its harmless characteristic to humans and robust survivability,MS2 (a bacteriophage that only parasitizes male Escherichia coli (E.coli) bacteria (Davis et al., 1961) and has an approximate particle sizeof 28 nm) is widely used as a surrogate in research of small airborneviruses and enteric viruses (Dawson et al., 2005; Zuo et al., 2014). InPhase 2 of this study, MS2 (#15597-131, ATCC®, Manassas, Va., USA) wasused as the challenge virus aerosol. The viability of MS2 bacteriophagein the BASIC was studied by shaking the bag to collect the amplifiedvirus aerosol into the water medium in the expansion bag. A single-layervirus plaque assay (VPA) technique was applied to the collected mediumfollowing the standard operating procedures provided by US EnvironmentalProtection Agency (USEPA, 1984).

Lyophilized MS2 bacteriophage was diluted in 100 mL DI water to make astock suspension with a titer of around 10¹¹ PFU/mL and stored in arefrigerator under 4° C. Prior to use, 1 mL MS2 stock suspension waspipetted and diluted into 100 mL DI water to create a titer of 10⁹PFU/mL. E. coli (#15597, ATCC®, Manassas, Va., USA) was used in VPA asthe indicator host cells for MS2 bacteriophage. E. coli powder wasaseptically inoculated onto a tryptone yeast extract agar (TYA) plateovernight, and then a single uniform colony on the plate was asepticallypicked and inoculated into sterile tryptone yeast extract broth-1(TYB-1), and incubated overnight to create an E. coli stock suspension.Prior to each experiment, 1 mL E. coli stock suspension was cultivatedin 30 mL TYB-1 for 6 h to obtain log phase cells of an appropriateconcentration. All incubations were held at 37° C.

In the single-layer VPA method, TYA was adopted as the plaque assaymedium. TYA contained 1.0 g tryptone, 0.1 g yeast extract, 0.1 gglucose, 0.8 g sodium chloride (NaCl), and 0.022 g calcium chloride(CaCl₂) per 100 mL of medium with 1.0 g additional agar. Tryptone yeastextract broth Type 2 (TYB-2) with all ingredients in TYA except agar wasmade for dilution of the samples. TYB-1 that contained only tryptone,yeast extract and sodium chloride were also used for cultivating E.coli, 6 h prior to each experiment.

Proper dilution was conducted for samples collected from the expansionbag. Preliminary tests determined that the dilution factors should be 1and 10 (i.e., the original and 1/10 factors were adopted for theassays). The agar was kept in a warm water bath at ˜50° C. to maintainits fluidity. Nine mL TYA, 1 mL serial diluted sample and 0.5 mL E. coliTYB-1 solution were mixed, vortexed and then poured into a Petri dishand gently shaken for spreading the agar evenly. Afterwards, the Petridish was placed bottom-up in an incubator at 37° C. where agar couldsolidified.

After the overnight incubation, viable virus lysed E. coli cells andplaques appeared on the bottom of the Petri dish. Only Petri dishes thatcontained 10-100 plaques were used for counting plaque forming unit(PFU) in order to provide an accurate count (Cormier & Janes, 2014). Bymultiplying PFU with the dilution factor, the titer of the viable MS2C_(viable) (PFU/mL) was determined, using Eq. (3).

$\begin{matrix}{C_{viable} = \frac{{PFU}_{{on}\mspace{14mu}{plate}} \times {DF}}{V}} & (3)\end{matrix}$where DF is the dilution factor and V is the volume of the dilutedsample.Sensitivity Analyses

Compression pressure, number of C/E cycles and water temperature werevaried for sensitivity analyses of the physical size amplification,while dwell time was also included for evaluation of the viabilitypreservation. Sensitivity analyses were carried out by the experimentaldesign shown in FIG. 30 with the baseline set of variables being 103.5kPa of compression pressure, one C/E cycle and 25° C. of DI watertemperature in physical size amplification tests. No uniform baselineset of values was set in the viability preservation tests. Instead, eachexperimental run was paired with a control group (no application of anyC/E cycle) and performed on the same day (due to the high variability ofthe results of VPA test on different days).

The VPA method was conducted on the samples according to the groupwherein the sensitivity analysis was conducted. The viable MS2 titer ofeach sample was then calculated and compared for viability preservationassessment. In order to monitor the stability of MS2 viability in theCollison nebulizer, the titer of viable MS2 in the nebulizer reservoirwas also measured for each group. An additional experiment to estimatethe rate of aerosol generation was conducted by monitoring the liquidvolume remained in the Collison nebulizer at different times; theconsumption rate of MS2 suspension was determined using the slope of thelinear regression of the data points. The total count of viable MS2 inthe expansion bag fed by the nebulizer reservoir was determined usingEq. (4), assuming no loss while transporting and nebulizing.A _(viable)(nebulizer)=C _(viable)(nebulizer)×CR×t  (4)where A_(viable) is the count of viable MS2 consumed in the reservoir(PFU), CR is the consumption rate (mL/min), C_(viable) (nebulizer) isthe titer of viable MS2 in the Collison nebulizer reservoir (PFU/mL),and t is the sampling time (10 s in all experiments). Quality Controland Data Analysis

Prior to each experiment, the aerosol generation system was stabilizedfor 15 min to ensure the variations of the flow rate within ±0.1 Lpm.Since aerosol size enlargement is realized through water vaporcondensation, relative humidity of the incoming aerosol stream should beminimal. Measurement of relative humidity before and after the diffusiondryer showed the relative humidity averagely decreased from ˜80% to˜35%. After each experiment, the bag was rinsed by 70% isopropyl alcoholand DI water. Ten mL of DI water was then poured into the bag as thecondensing medium right before the next experiment, and the temperatureof the DI water was immediately measured by an Infrared Thermometer(Etekcity® Co. Ltd., Anaheim, Calif., USA). The expansion bag was thensealed with a lid and held to the chamber to be vacuumed. In viabilityevaluation experiments, all test tubes and solutions were autoclaved at120° C. and 1 atm for at least 30 min after each experimental run.

It should be noted that maintaining the water temperature at the highesttested temperature of 60° C. from the time it was poured into theexpansion bag to when the adiabatic expansion was applied, waschallenging. Based on our measurements right after application of oneC/E cycle, temperature of the DI water dropped from 60° C. to 40° C.(%50) for the experimental run of 60° C., and dropped from 40° C. to 35°C. (%12.5) for the experimental run of 40° C. In other word, due to thetemperature decrease of the control volume caused by adiabaticexpansion, DI water temperature could not maintain its original value,and the temperature drop was larger at the higher initial temperature.

To assess the statistical validity, each experimental condition wastriplicated. To analyze the data obtained from the BASIC, a 2-tailedt-test for unequal variance was implemented for comparing thestatistical significances between the baseline group and the controlgroup. One-way analysis of variance (ANOVA) was applied for in-groupcomparison and a post-hoc test using Bonferroni's method was applied forcomparison of two subgroups within a group.

Results and Discussion

Physical Size Amplification

Compression Pressure

Size distributions of the aerosol with different compression pressuresand without adiabatic expansion are displayed in FIG. 25. FIG. 25 showsthe physical size distributions of MS2 aerosol (i.e. concentration as afunction of particle size) amplified by the adiabatic expansion process.The measurement was done using an Optical Particle Counter that measuressizes of 0.3 μm and above. The source aerosol was mainly smaller than0.3 μm (hence only a small fraction above 0.3 μm was detected). As thecompression pressure for inducing adiabatic expansion increased from 69kPa to 103.5 kPa to 138 kPa, the number of supermicron (<1 μm) aerosolparticles increased. The axes are in log-scale. As shown, a fraction ofsubmicron (0.3-1.0 μm) particles picked up some moisture content andwere amplified slightly even in the absence of a C/E cycle. In thebaseline group, after application of only one C/E cycle at thecompression pressure of 103.5 kPa, anincrease of particles was observed:the largest particle size detected after one C/E cycle was ˜4 μm, andthe number concentration of supermicron particles compared to the sourceaerosol was very high (>300#/cm³). An increase in compression pressureresulted in an improved size amplification performance, and the numberconcentration increased further. When compression pressure increasedfrom 69.0 kPa to 103.5 kPa, the number concentration increased 4 to 5times in the submicron size range, and the largest particle sizeincreased from 2.5 μm to >4 μm. At compression pressure of 138.0 kPa,the largest size of amplified particles exceeded 5 μm and a very obviousincrease of number concentration in the supermicron range took place.

The t-test results of number concentration of the supermicron particlesbetween groups with (control group) and without adiabatic expansion(baseline group) are displayed in FIG. 31. The results confirmed thatthere was a difference (P-value <0.0001) between the two groups. T-teston CMD of supermicron particles also proved that adiabatic expansionenlarged particles. Although the fraction of particles larger than 3 μmwas negligible, this does not necessarily mean that 3 μm was theupper-limit of the particle enlargement. Due to gravitational settling,larger particles might have settled (on the inner surface of theexpansion bag or tubing) before reaching the OPC. In addition, theinstant expansion process causes turbulence inside the bag, which isconducive to the deposition of larger particles by impaction (Robertson& Goldreich, 2012). In other words, there may be constraints in thecurrent system for accurate measurement of aerosol particles larger thana few microns.

One-way ANOVA results on compression pressures are displayed in FIGS.26(a)-26(b). An increase in the number concentration of the supermicronparticles with an increase in compression pressure was observed. A10-fold increase in number concentration was achieved by increasing thecompression pressure, for example from 69.0 kPa to 138.0 kPa (FIG.26(a)). CMD also increased from ˜1.48 μm to ˜2.17 μm (FIG. 26(b)). Ahigher compression pressure can provide a higher supersaturation ratio,leading to a lower temperature after the C/E cycle. The lowertemperature can enable more water vapor condensation onto the particles,thus a higher number of the nanosized particles undergoing the sizeamplification.

Number of C/E Cycles

One-Way ANOVA test results as shown in FIGS. 27(a)-27(b) reveal nostatistically significant difference in number concentration or CMDwithin the three groups (p-value ˜0.21). After the first C/E cycle,thermodynamic equilibrium inside the bag may have been reached. Whilemore C/E cycles were originally hypothesized to provide more water vaporfor size amplification, the experimental results indicate that when thenext cycle of compression applied, work was done to the aerosol, causingre-evaporation of the water from the amplified aerosol in the bag. Thus,a higher number of the C/E cycles simply repeated the first cycle andexerted no observed net effect on amplification.

DI Water Temperature

As shown in FIG. 28(a), an increase in the number concentration wasobserved when the DI water temperature increased from 25° C. to 40° C.However, the number concentration for the size range of study decreasedwhen the DI water temperature increased further to 60° C. The results of1-Way ANOVA test also confirm that the experiment run at 40° C. had thegreatest size amplification potency among these three temperatures. Noobvious difference among the CMD of these three groups is seen (FIG.28(b)). A higher temperature was expected to produce more supermicronparticles as it would yield a higher moisture content available forcondensation. One possible reason for a lower number concentration at60° C. is that at this higher temperature, the stability of virus capsidmay have reduced, which might have led to the structure alteration ofvirion protein (a complete virus particle) and finally affected thehydrophobicity for MS2 aerosol (Pinto et al., 2010).

Viability Preservation

The MS2 titer in the Collison nebulizer reservoir was 1.5 (±0.32)×10⁹PFU/mL, thereby implying the system supplied a stable size distributionof the aerosol source for different experiments. The consumption rate ofMS2 suspension in the Collison nebulizer was about 0.3 mL/min, and thebag filling time was set at 10 s for each experiment. Consequently, theconsumed volume of the nebulizer liquid was about 0.05 mL, andaccordingly the expected MS2 titer in the sampling air was estimated tobe ˜7.5×10⁷ PFU/L of air, assuming no loss due to transport orinactivation by the nebulization process. Detailed results for eachsystem parameter investigated are reported in the followingsub-sections.

Compression Pressure

Results of the statistical analyses are displayed in FIG. 29(a), whichshows the viable MS2 aerosol collected after going through the adiabaticprocess for size amplification. FIG. 29(a) displays the viable MS2 titercollected as a function of compression pressure (for inducing adiabaticexpansion). As the pressure increased, the viable count increased. FIG.29(c) illustrates one compression/expansion cycle to be the optimalcondition. FIG. 29(c) illustrates that the viable count increased astemperature increased, although at high temperature (60° C.) some MS2got deactivated. FIG. 29(d) shows that the amplification process wasvery fast and additional dwell time was not needed to enhance theperformance.

FIG. 29(a), shows an increasing trend of collected viable MS2 with anincrease in the compression pressure up to 103.5 kPa. Shaking amplifiedaerosol trapped inside the expansion bag increased the total gain of theaerosol and probability of virus deposition into the DI water. Theresults indicate the viability was preserved well in the range ofcompression pressure investigated (69.0-138.0 kPa). Past studies showedthat application of a high pressure on a virus-contained suspension cankill noroviruses (Aertsen et al., 2009). However, this concern appliesto an extremely high pressure (>60,000 psi) wherein virus capsid proteinor lipid envelope may break (Tang et al., 2010). By using MS2 as asurrogate at a high pressure (40,000 psi), Pan (2015) observed that MS2did not suffer significant viability loss under that pressure held for 3min. Our statistical analysis results infer that the collection ofviable MS2 after adiabatic expansion achieved a statisticallysignificant increase than the case without adiabatic expansion, and thecollected amount increased as compression pressure increased up to 103.5kPa. There was no statistically significant difference between caseswith compression pressures of 103.5 kPa and of 138.0 kPa.

Number of C/E Cycles

Comparative results of collected viable MS2 under different numbers ofC/E cycles are presented in FIG. 29(b). In contrast to the physical sizeamplification, a higher number of cycles may be inversely related to theviability preservation of MS2. Pollard (1960) reported that gradualexertion of pressure onto viruses leads to an increase of bond stabilityin virion protein, thus leading to the better stabilization of virus.However, exertion of an instant pressure loss (as in adiabaticexpansion) may cause viruses to lose the antigenic surface and breakapart. In other words, when the number of C/E cycles increases, virusesmay suffer from frequent and instant pressure changes, which can causevirions to expand and break apart. As seen in FIG. 29(b), the highestviable virus titer (4620±930 PFU/L air) was achieved at one C/E cycle,and the difference from other groups (3 cycles, 5 cycles, and noadiabatic expansion) was statistically significant. Taking the resultsof compression pressure together into consideration, it can be concludedthat MS2 may have a high endurance to pressure, yet low resistance tofrequent pressure change. Hence, when using the BASIC for collection ofviable MS2, one C/E cycle can provide the optimal outcome.

DI Water Temperature

Collected viable MS2 as a function of the temperature of pre-injectedwater is plotted in FIG. 29(c). In operating the BASIC, DI water wasused as a medium for both condensation and collection, although othermedia may be used. When applying DI water of 40° C., an increase ofviable MS2 collected in water was observed in comparison to the casewith DI water of 25° C. However, when the water temperature increasedfurther to 60° C., the titer of viable MS2 collected in water decreasedgreatly. This was likely due to inactivation of MS2 by hot water beyondthe comfort zone of MS2. Previous studies found that temperature canplay a vital role in the viability of many viruses (Ausar et al., 2006).Pinto et al. (2010) investigated the effect of temperature on MS2, andreported that high temperatures can cause structural change of virioncapsid and therefore virus inactivation. One-way ANOVA results confirmthat by applying water temperature of 40° C., the titer of collectedviable MS2 was higher than those at 25° C. and 60° C. Different fromother key parameters, higher water temperature may exert positive effecton physical size amplification but negative effect on viabilitypreservation. As MS2 shows vulnerability to 60° C., therefore 40° C. isthe optimal DI water temperature, considering both physical collectionand viability preservation.

Dwell Time

There was no benefit for increasing the dwell time since the viable MS2titer did not vary much among the four dwell times studied (see FIG.29(d)). This is confirmed by the 1-Way ANOVA test showing nostatistically significant difference among the four dwell time groups.C.-Y. Wu and Biswas (1998) reported that the growth of an aerosolparticle in the free molecular regime due to condensation wasindependent of its original size, and it could achieve 50% of its finalsize at ˜3 characteristic times. When the number concentration ofultrafine particles was above 10⁴#/cm³, the characteristic time was lessthan 0.3 s. In other words, in a short period of time (less than 1 s),ultrafine particles could grow to 50% of its final size (calculated as˜10 μm). Hence, a long dwell time for particle growth may not beessential (neither for particle amplification nor for more viruscollection in this given system).

Comparison with SKC® BioSampler

The SKC® BioSampler is a commercially available sampler commonly used inbioaerosol studies. Many studies have illustrated its good efficiencyfor collecting supermicron particles, e.g., bacteria and fungi (Kesavanet al., 2010; Lin et al., 1999; Y. Wu et al., 2010), although itsefficiency for virus aerosols below 100 nm is unsatisfactory (<10%)(Hogan, et al., 2005). Fabian et al. (2009) used a titer of 1.9×10¹⁰ FFU(virus focus forming unit)/mL in the original suspension of influenzavirus in the nebulizer reservoir and achieved the titer of ˜400 FFU/Lair in collected sample from the BioSampler. Pan et al. (2016) also usedMS2 as test virus aerosol, and obtained a result of only ˜10 PFU/L airfrom the BioSampler by using an original titer of 10⁹ PFU/mL in theCollison nebulizer. Compared to these results, the BASIC has achieved upto >4,000 PFU/L air under the optimal conditions (i.e. compressionpressure of 138.0 kPa, 1 C/E cycle and DI water temperature of 40° C.;no additional dwell time applied).

Transport and nebulization can cause a major viability loss of producedMS2 aerosol. (Thompson and Yates (1999)) reported that MS2 suffered agreat viability loss when a triple-phase-boundary (TPB, the interface ofgas, liquid and solid) existed. The shear force established in thenebulization process can easily damage the viruses in the TPB. Asmentioned before, the titer of MS2 in the produced aerosol was estimatedto be ˜7.5×10⁷ PFU/L air. Thus, based on the measured MS2 titer in theCollison nebulizer reservoir, the collection efficiency in the BASIC wasonly ˜0.005%. However, it is still much higher when compared with thecollection efficiency of the BioSampler (<0.0001%) (Pan, et al., 2016);the BASIC can collect 50 times more under optimal conditions over theBioSampler.

CONCLUSIONS

The present example focused on the performance assessment of the BASICin enabling ultrafine virus aerosol sampling as well as the ability inpreserving the viability of airborne virus. MS2 phage was used as thetest agent in assessing both amplification effectiveness and viabilitypreservation. Results for physical size amplification tests showed thatincreasing compression pressure in the range of 69.0-138.0 kPa had apositive effect on the CMD enlargement and increase of aerosol numberconcentration. This can be attributed to the higher saturation ratio athigher compression pressure. The application of C/E cycles yieldedphysical size amplification, and 3 cycles can be the optimal condition.Water temperature had a double-edged effect on MS2 aerosol. Increasingwater temperature from 25° C. to 40° C. resulted in a positive effect,but it exhibited a negative effect as temperature increased from 40° C.to 60° C. This phenomenon might be due to the structural change ofcapsid protein of MS2 virion, which reduced MS2's ability to attractwater vapor.

In evaluating the performance of viability preservation, the resultsshowed that increasing compression pressure also produced an improvementin the total amount of collected MS2 in DI water, and the range ofcompression pressure applied in this study reflected a negligible effecton the viability of MS2 virus. Regarding the number of C/E cycles,applying one single cycle was can be optimal for collecting viable virusaerosol in the expansion bag as multiple C/E cycles induced a greatvirus viability loss. This reflected that MS2 might not be resistant tofrequent pressure swing. Similar to the results in the physical sizeamplification, 40° C. water temperature increased the amount ofcollected viable MS2 virus, while 60° C. water temperature sampled muchless. The results are in agreement with previous research wherein hightemperature above a threshold value could alter the structure of viruscapsid protein, leading to inactivation of viruses. Increasing dwelltime from 0 s to 120 s yielded little to no obvious difference in thetiter of viable MS2.

In conclusion, the BASIC system showed its potential in highly efficientsampling of ultrafine virus aerosols. When the BASIC is combined with arapid airborne virus detection and analysis approaches, the resultingsystem could be a significantly improved virus aerosol detection andidentification system for infection control, agriculture, research, andbiodefense applications.

REFERENCES

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It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range. In an embodiment, the term “about” can includetraditional rounding according to the values and/or measuringtechniques. In addition, the phrase “about ‘x’ to ‘y’” includes “about‘x’ to about ‘y’”.

Many variations and modifications may be made to the above-describedembodiments. All such modifications and variations are intended to beincluded herein within the scope of this disclosure and protected by thefollowing claims.

At least the following is claimed:
 1. A bioaerosol amplification anddetection system, comprising: a bioaerosol amplification unit comprisinga chamber configured to receive air containing bioaerosols, wherein thechamber is further configured for adiabatic amplification of thebioaerosols; and a biosampler, wherein the biosampler is in fluidcommunication with the bioaerosol amplification unit, wherein thebiosampler is configured to receive and collect adiabatically amplifiedbioaerosols from the chamber of the bioaerosol amplification unit; andwherein the air containing bioaerosols is cooled by a temperature dropwithin the chamber of the bioaerosol amplification unit, wherein thetemperature drop is controlled by the ratio of the pressure of the aircontaining bioaerosols after adiabatic expansion to the pressure of theair containing bioaerosols before adiabatic expansion.
 2. The bioaerosolamplification and detection system of claim 1, wherein the adiabaticamplification is adiabatic cooling.
 3. The bioaerosol amplification anddetection system of claim 2, wherein the adiabatic cooling furthercomprises swirling, mixing, or both.
 4. The bioaerosol amplification anddetection system of claim 1, further comprising a bioaerosol analysisplatform, wherein the bioaerosol analysis platform is configured toreceive adiabatically amplified bioaerols collected in the biosamplerand configured to detect the adiabatically amplified bioaerosols by oneor more bioaerosol detection assays.
 5. The bioaerosol amplification anddetection system of claim 4, wherein the bioaerosol analysis platform isa microfluidic device comprising one or more bioaerosol detection assaysconfigured to detect the adiabatically amplified bioaerosols.
 6. Thebioaerosol amplification and detection system of claim 1, wherein thebioaerosol amplification unit further comprises one or more interiorsurfaces of the chamber wetted with water having a temperature of about35° C. to about 65° C., wherein the one or more surfaces is adjacent tothe air containing bioaerosols.
 7. The bioaerosol amplification anddetection system of claim 1, wherein the chamber further comprisescooled air containing bioaerosols having a temperature of about −40° C.to about 10° C. and steam having a temperature of about 35° C. to about65° C.
 8. The bioaerosol amplification and detection system of claim 1,wherein the chamber further comprises cooled air having a flow rate ofabout 0.1 Liters/min to about 10 Liters/min and steam having a flow rateof about 1 Liters/min to about 50 Liters/min.
 9. The bioaerosolamplification and detection system of claim 4, wherein the microfluidicdevice is paper-based or laminated paper-based.
 10. The bioaerosolamplification and detection system of claim 4, wherein the one or moredetection assays comprise an immunoassay or a nucleic acid amplificationassay, individually or in combination.
 11. The bioaerosol amplificationand detection system of claim 4, wherein the bioaerosol amplificationand detection system is configured to detect viruses.
 12. A method ofdetecting amplified bioaerosols, comprising the steps of: providing abioaerosol amplification and detection system comprising a bioaerosolamplification unit, a biosampler, and a bioaerosol analysis platform;delivering air containing bioaerosols to the bioaerosol amplificationunit, wherein the bioaerosol amplification unit is configured toadiabatically amplify bioaerosols; adiabatically amplifying bioaerosolswith the bioaerosol amplification unit; delivering amplified bioaerosolsfrom the bioaerosol amplification unit to the biosampler; precipitating,concentrating, or both the amplified bioaerosols into a collectionreservoir of the biosampler; delivering the collected amplifiedbioaerosols from the collection reservoir of the biosampler to abioaerosol analysis platform, wherein the bioaerosol analysis platformis configured to detect one or more collected amplified bioaerosols orcomponents thereof with one or more detection assays; detectingcollected amplified bioaerosols or bioaerosol components with one ormore detection assays; and wherein the air containing bioaerosols iscooled by a temperature drop within the bioaerosol amplification unit,wherein the temperature drop is controlled by the ratio of the pressureof the air containing bioaerosols after adiabatic expansion to thepressure of the air containing bioaerosols before adiabatic expansion.13. The method of claim 12, wherein the one or more detection assays isa nucleic acid detection assay or an immunoassay, individually or incombination.
 14. The method of claim 12, wherein the one or moredetection assays is configured to detect one or more viruses.
 15. Themethod of claim 12, wherein the collection reservoir of the biosamplerfurther comprises a collection media.
 16. The method of claim 12,wherein the air containing bioaerosols is cooled within a chamber of thebioaerosol amplification unit, wherein the chamber of the bioaerosolamplification unit has one or more interior surfaces adjacent to the aircontaining bioaerosols, wherein the one or more surfaces are wetted withwarm water.
 17. The bioaerosol amplification and detection system ofclaim 1, wherein the chamber is configured so that the volume of thechamber can be reduced by compression and expanded by decompression. 18.The bioaerosol amplification and detection system of claim 1, whereinthe biosampler is functionally integrated into the chamber of thebioaerosol amplification unit and the chamber is configured forcollection of amplified bioaerosols.